Methods, Systems, And Compositions For Determining Blood Clot Formation, And Uses Thereof

ABSTRACT

A method is directed to determining a thrombosis function and includes flowing a fluid sample over a surface having a fixed endothelial cell monolayer. The method further includes stimulating the fixed endothelial cell monolayer to induce formation of a clot, the clot being formed via interaction between the fixed endothelial cell monolayer and the fluid sample. In response to the clot formation, the method further includes determining a thrombosis function associated with the fluid sample and the fixed endothelial cell monolayer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and benefit of U.S. ProvisionalPatent Application Ser. No. 62/165,272, filed on May 22, 2015, and U.S.Provisional Patent Application Ser. No. 62/310,166, filed on Mar. 18,2016, each of which is hereby incorporated by reference herein in itsentirety.

GOVERNMENT SUPPORT

The invention was made with Government Support under N66001-11-1-4180awarded by the Space and Naval Warfare Systems Center of the U.S.Department of Defense, and under HR0011-13-C-0025 awarded by the DefenseAdvanced Research Projects Agency of the U.S. Department of Defense. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates generally to quantifying athrombosis-related function in vitro based on physiologically relevantconditions, and, more particularly, to a microfluidic system havingfluid flow interaction between a fixed endothelial layer and cells (suchas platelets) in a fluid sample.

BACKGROUND OF THE INVENTION

Generally, the vascular endothelium and shear stress are criticaldeterminants of hemostasis and platelet function in vivo, and yet,current diagnostic and monitoring devices do not fully incorporateendothelial function under flow in their assessment. Therefore, currentdiagnostic and monitoring devices can be unreliable and inaccurate.Furthermore, it is challenging to include the endothelium in assays forclinical laboratories or point-of-care settings because living cellcultures are not sufficiently robust.

More specifically, mutual signaling between endothelium and activatedplatelets is widely recognized as critical for regulation of hemostasisand thrombotic disorders associated with various diseases, includingatherosclerosis, sepsis, and diabetes. Yet, no practical diagnosticassays exist that can measure cross-talk between platelets and inflamedvessel walls in the presence of physiological shear. Over the lastdecade or so, multiple flow chambers and microfluidic devices thatcontain microchannels have been lined by living endothelium and exposedto flowing blood to study the basic science of thrombosis. While thesedevices have been very useful in advancing research, they have not beenused in clinical settings due to the difficulty in maintaining livingendothelial cells in them. Specifically, because it is extremelydifficult to maintain the viability of living cell cultures for extendedtimes in non-controlled settings, it is virtually impossible to rely onthese assays. Therefore, the only microfluidic devices that arecurrently being deployed in clinical diagnostic settings are lined withcollagen to mimic thrombus formation and platelet aggregation induced inresponse to vascular injury, and, thus, they fail to capture thephysiological interplay between endothelial cells, platelets and fluidshear stress that is so relevant to hemostasis in inflammatory diseases.

Additionally, pulmonary microvascular thrombosis is a catastrophiccondition amounting to a large number of patient deaths worldwide.Despite significant progress in understanding fundamental biology oflung hemostasis and thrombosis, it is still very difficult to predictresponse and study mechanism of action of potential drug candidates tohumans. This is partly so because currently available in vitro assays donot recapitulate physiologically-relevant forces, such as shear stress,and animal models can be very complex allowing limited experimentalmanipulation, making it impossible to dissect and study intercellularsignaling.

More specifically, pulmonary intravascular thrombosis and plateletactivation initiating from, for example, acute lung injury (“ALI”),acute chest syndrome (“ACS”), pulmonary hypertension (“PH”), chronicobstructive pulmonary disease (“COPD”), and acute respiratory distresssyndrome (“ARDS”), are causes of significantly high patient mortalityand morbidity. Therefore, pulmonary intravascular thrombosis andplatelet activation are also promising and emerging therapeutic targetsto save and prolong patient life. Although epithelial injury,endothelial dysfunction, and in situ thrombotic lesions are observedoften in human patients in chronic pulmonary diseases, animal models ofpulmonary dysfunction are still unable to completely mimic the alteredhemostasis and hemodynamic complexity of the lung. Importantly, animalmodels can be very complex and it may be impossible to study cell-cellinteractions between multiple tissues independently of each other duringblood clotting or drug administration. Based on this type oflimitations, along with ethical barriers associated with animal models,it is desirable to advance in vitro disease models of pulmonarythrombosis that can mimic human organ-level functionality and complementor reduce reliance on animal studies, to enable more reliable basicresearch and make drug discovery more efficient.

In vitro, commercially available coagulation and platelet functiontechnologies also have serious limitations due to the fact that they donot incorporate physiological tissue-tissue or cell-cell interactions,and relevant fluid dynamics of blood cells, which are key determinantsof thrombosis. In research laboratories, dishes and transwell plateshave been used for decades to culture cells and study basic biology, butthese are static systems, highly non-physiological and cannotrecapitulate tissue or organ-level functionality. For example, this typeof systems cannot recapitulate blood flow or breathing of a lung.

To incorporate blood perfusion, parallel plate-flow chambers have beenwidely applied in the past three decades or so to measure thrombusformation and platelet adhesion kinetics. However, being macroscaledevices, these chambers do not mimic small blood vessels, typically donot incorporate endothelium, and require large blood sample volumes foranalysis.

More recently, microfluidic devices lined with human endothelial cellshave shown that endothelial activation, platelet adhesion and fibrinformation in the presence of physiological shear can be somewhatvisualized. However, these devices are also limited in studyingorgan-level pulmonary thrombosis, in part because they do not includethe role of live epithelial cells, dynamic platelet-endothelialinteractions (e.g., activation, aggregation, adhesion, translocation,and embolization) in the lumen that occur over large spatiotemporalscales, and often do not incorporate perfusion of whole blood.

Recently, microfluidic technology has been advanced to demonstrate anorgan-level in vitro model of a lung and pulmonary edema, where alveolarepithelial and endothelial cells were co-cultured in two overlayingchambers, respectively. Fibrin formation in the alveolar chamber wasanalyzed in the presence of an inflammatory cytokine IL-2 and in thepresence of flow and relevant cyclic stretch. However, this type oflung-on-a-chip model still lacks relevant functionality for mimickingrelevant foundational conditions of pulmonary thrombosis. For example,the endothelial chamber only contains one side cultured with the cellsand hence, it does not contain an endothelial lumen. Based on thislimitation, the device is not appropriate for perfusing whole blood andfor studying blood cell-endothelial interactions. In fact, other than adilute suspension of neutrophils, none of the blood cells or plateletshas been perfused or analyzed in this type of device, in itsphysiological concentration.

Another limitation of the long-on-a-chip model is that it usesnon-primary epithelial cell lines, A549 or NCI-H441. Although this typeof model mimics certain aspects of human lung function, it is not idealin the context of mimicking physiologically-relevant hemostasis andthrombosis, as they are derived from tumors and, therefore, canpotentially alter endothelial and platelet function.

Therefore, there is a continuing need for solving the above and otherproblems.

SUMMARY OF THE INVENTION

According to one aspect of the present invention, a microfluidic deviceis lined with a human endothelium that is chemically fixed, but stillretains its ability to modulate hemostasis under continuous flow invitro. For example, according to one method, microfluidic channels areseeded with collagen and endothelial cells and are left either untreatedor treated with tumor necrosis factor-α (TNF-α). The cells are, then,fixed with formaldehyde. Recalcified citrated whole blood (0.5 mL) fromhealthy volunteers or patients taking antiplatelet medication isperfused and platelet coverage is recorded. The chemopreservedendothelialized device is lined with a bioinspired material thatsupports formation of platelet-rich thrombi in the presence ofphysiological shear, similar to a living arterial vessel. Furthermore,the method demonstrates the potential clinical value of thechemopreserved endothelialized device by showing that thrombus formationand platelet function are measurable within minutes using a small volumeof whole blood taken from subjects receiving antiplatelet medications.The method further demonstrates potentially greater reliability thanstandard platelet function tests and collagen-coated perfusion chambers.

According to another aspect of the present invention, a microengineeredlung-on-chip device is used for studying human pulmonary blood clottingand platelet-endothelial interaction dynamics. The lung-on-chip is amicrofluidic device populated with primary alveolar cells (“AE”)localized within a top channel and vascular endothelial cells in abottom compartment. The top channel and the bottom compartment areseparated by a matrix-coated membrane. Whole blood is perfused in thevascular compartment while the epithelium is stimulated with a cytokineor endotoxin, and platelet-endothelial interactions are recorded inreal-time. To quantify the dynamics of the platelet-endothelialinteractions, a stochastic analytical method is provided that is highlysensitive to changes in endothelial and platelet activation. In vitro,the presence of alveolar epithelium is shown to be beneficial forreconstituting pulmonary thrombosis in response to an inflammatorystimulus of lipopolysaccharide (“LPS”). Additionally, this model is usedin drug development by analyzing the effect of a novel proteaseactivator receptor-1 (“PAR1”) antithrombotic compound, termed parmodulin2 (“PM2”), and demonstrate that PM2 has an endothelial cytoprotectiveeffect in response to LPS-mediated inflammation. The lung-on-chip devicereconstitutes organ-level functionality that accurately reflects manyaspects of human pulmonary thrombosis and appears to offer a valuableplatform for drug development.

According to one aspect of the present invention, a method is directedto determining a thrombosis function and includes flowing a fluid sampleover a surface having a fixed endothelial cell monolayer. The methodfurther includes stimulating the fixed endothelial cell monolayer toinduce formation of a clot, the clot being formed via interactionbetween the fixed endothelial cell monolayer and the fluid sample. Inresponse to the clot formation, the method further includes determininga thrombosis function associated with the fluid sample and the fixedendothelial cell monolayer.

According to another aspect of the invention, a microfluidic system isdirected to determining a thrombosis function. The microfluidic systemincludes a compartment having a surface with a fixed endothelial cellmonolayer, the compartment being configured to receive a fluid sampleflowing over the surface such that cells in the fluid sample interactwith the fixed endothelial cell monolayer. The microfluidic systemfurther includes a detection module configured to detect interactionbetween the cells and the fixed endothelial cell monolayer, and todetermine a function of the cells in the fluid sample.

According to yet another aspect of the invention, a device is directedto simulating a function of a tissue. The device includes a firststructure defining a first microchannel and configured to have a fluidsample flowing within, the fluid sample including platelets. The devicefurther includes a second structure defining a second microchannel, anda membrane located at an interface region between the first microchanneland the second microchannel. The membrane has a first side facing towardthe first microchannel and a second side facing toward the secondmicrochannel, the membrane separating the first microchannel from thesecond microchannel. The first side of the membrane includes a fixedendothelial cell monolayer, the second side of the membrane including atleast one layer of tissue-specific cells. The device further includes adetection module configured to detect interaction between the plateletsand the fixed endothelial cell monolayer. The detection module isfurther configured to determine a function of the platelets in the fluidsample.

According to yet another aspect of the invention, a system is directedto quantifying thrombosis in vitro based on physiological conditions.The system includes a solid substrate having a surface with a fixedendothelial cell monolayer, and a detection module configured to receivethe solid substrate. The detection module is further configured todetect spatial and temporal interaction between cells in a fluid sampleand the surface of the solid substrate when the fluid sample is flowedover the surface along a flow axis. The system further includes one ormore controllers configured to store time-lapse data of detectablesignals collected from the detection module, wherein the detectablesignals represent spatial and temporal interaction between the cells andthe surface.

The one or more controllers are further configured to generate akymograph from at least a portion of the stored time-lapse data, whereina time axis of the kymograph indicates at least a portion of thetime-lapse duration, a space axis of the kymograph indicating thedetectable signals along the flow axis. The one or more controllers arefurther, yet, configured to determine, based on the generated kymograph,a rate of fluctuation in a coefficient of variation (CV) of thedetectable signals to generate a temporal cell dynamics index, and todetermine either (i) the presence of reactive cells in the fluid samplewhen the temporal cell dynamics index is higher than a temporal controlvalue, or (ii) the absence of reactive cells in the fluid sample whenthe temporal cell dynamics index is no more than the temporal controlvalue. The system further includes a display module for displayingcontent that is based in part on output determined by the one or morecontrollers, wherein the content includes a signal indicative of eitherpresence or absence of at least one of reactive cells or cellaggregation in the fluid sample.

According to yet another aspect of the invention, a method is directedto quantifying thrombosis in vitro based on physiological conditions.The method includes providing a solid substrate having a surface with afixed endothelial cell monolayer, and detecting, via a detection module,spatial and temporal interaction between cells in a fluid sample and thesurface of the solid substrate when the fluid sample is flowed over thesurface along a flow axis. The method further includes storing, via oneor more controllers, time-lapse data of detectable signals that arecollected from the detection module, the detectable signals representingspatial and temporal interaction between the cells and the surface. Themethod also includes generating a kymograph, via at least one of the oneor more controllers, from at least a portion of the stored time-lapsedata, a time axis of the kymograph indicating at least a portion of thetime-lapse duration, a space axis of the kymograph indicating thedetectable signals along the flow axis.

Based on the generated kymograph, the method determines, via at leastone of the more controllers, a rate of fluctuation in a coefficient ofvariation (CVO) of the detectable signals to generate a temporal celldynamics index. The method further includes determining, via at leastone of the one or more controllers, (i) the presence of reactive cellsin the fluid sample when the temporal cell dynamics index is higher thana temporal control value, or (ii) the absence of reactive cells in thefluid sample when the temporal cell dynamics index is no more than thetemporal control value. The method further includes displaying, via adisplay module, content that is based in part on output determined bythe one or more controllers, the content including a signal indicativeof either presence or absence of at least one reactive cells or cellaggregation in the fluid sample.

According to yet another aspect of the invention, a system is directedto determining dynamics of platelets in a fluid sample. The systemincludes a solid substrate having a surface with a fixed endothelialcell monolayer, and a detection module configured to receive the solidsubstrate. The detection module is further configured to detect spatialand temporal interaction between cells in a fluid sample and the surfaceof the solid substrate when the fluid sample is flowed over the surfacealong a flow axis. The system further includes one or more controllersconfigured to store time-lapse data of detectable signals collected fromthe detection module, wherein the detectable signals represent spatialand temporal interaction between the cells and the surface.

The one or more controllers are further configured to generate akymograph from at least a portion of the stored time-lapse data, whereina time axis of the kymograph indicates at least a portion of thetime-lapse duration, a space axis of the kymograph indicating thedetectable signals along the flow axis. The one or more controllers arealso configured to determine, based on the generated kymograph, a rateof fluctuation in a coefficient of variation (CV) of the detectablesignals to generate a platelet dynamics index, the platelet dynamicsindex being one or more of a temporal platelet dynamics index and aspatial platelet dynamics index. The one or more controllers are furtherconfigured to determine either (i) the presence of reactive platelets inthe fluid sample when the platelet dynamics index is higher than acontrol value, or (ii) the absence of reactive platelets in the fluidsample when the platelet dynamics index is no more than the controlvalue. The system further includes a display module for displayingcontent that is based in part on output determined by the one or morecontrollers, wherein the content includes a signal indicative of eitherpresence or absence of at least one of reactive platelets or plateletaggregation in the fluid sample.

In addition, the inventors have shown that the fixed endothelial cellmonolayers that have been stored for a period of time (e.g., at leastabout 5 days or more) without freezing were still applicable forplatelet function analysis. Not only can this concept be applied toplatelet function analysis, but it can also be generally extended toanalyses of interaction dynamics of other cell types.

Further, instead of merely determining area-averaged platelet adhesion—astatic analysis—as regularly used in existing platelet functionassessment, the inventors have developed novel analytical methods toquantify temporal and/or spatial changes in the way of how cellsinteract with each other and/or to a surface. In some embodiments, theinventors have showed that the resulting characteristic temporal andspatial indices were sensitive enough to distinguish activated platelets(e.g., due to inflamed endothelial cells) and non-activated platelets.Thus, the temporal and spatial indices can be used as markers todiagnose diseases or disorders (e.g., platelet-associated disease ordisorder), to select appropriate therapy (e.g., anti-platelet and/oranti-inflammation therapy), to monitor treatment efficacy (e.g., toprevent recurrent thrombosis or bleeding), drug screening and/or todetermine drug toxicology. Accordingly, embodiments of various aspectsdescribed herein relate to methods, systems, and compositions fordetermining dynamic interaction of cells with each other, and/or withother cell types, and uses thereof.

One aspect described herein relates to a method of determining cellfunction. The method comprises (a) flowing a fluid sample over a surfacecomprising a monolayer of cells of a first type thereon; and (b)detecting interaction between cells of a second type in the fluid sampleand the monolayer of cells of the first type. The function of the cellsof the second type in the fluid sample can then be determined based onthe detected cell interaction.

In some embodiments, the fixed monolayer of cells of the first type cancomprise endothelial cells, and the cells of the second type in thefluid sample can comprise blood cells, e.g., platelets. Accordingly,another aspect provided herein relates to a method of determiningplatelet function, which comprises (a) flowing a fluid sample over asurface comprising a fixed endothelial cell monolayer thereon; and (b)detecting interaction between blood cells (e.g., platelets) in the fluidsample and the fixed endothelial cell monolayer.

In some embodiments, the fixed cell monolayer (e.g., fixed endothelialcell monolayer) can be derived from fixing target cell extract (e.g.,endothelial cell extract) and/or target cell-associated proteins (e.g.,endothelial cell-associated proteins) that are adhered to the surface.The target cell-associated proteins can comprise proteins secreted bythe target cells and/or present on the target cell surface. Where thetarget cell-associated proteins comprise endothelial cell-associatedproteins, examples of endothelial cell-associated proteins can include,but are not limited to, any art-recognized procoagulatory and/oranti-coagulatory proteins. In some embodiments, the endothelialcell-associated proteins can comprise von Willebrand factor and/ortissue factor (TF).

Any cell-comprising fluid sample can be flowed over the fixed cellmonolayer and it can vary depending on what target cells to be analyzed.In some embodiments, the fluid sample can comprise a blood sample, aserum sample, a plasma sample, a lipid solution, a nutrient medium, or acombination of two or more thereof. In some embodiments when the fluidsample comprises a blood sample, the method can further compriseremoving red blood cells from the blood sample prior to flowing theblood sample over the surface. In some embodiments, the fluid sampleflowing over the surface in the methods described herein can comprisecalcium ions and/or magnesium ions.

The surface over which the fluid sample flows can be a surface of anyfluid-flowing conduit disposed in a solid substrate that is compatibleto the fluid sample and the cells. In some embodiments, the solidsubstrate can comprise a cell culture chamber. For example, in oneembodiment, the surface can be a wall surface of a microchannel. In oneembodiment, the surface can be a surface of a membrane.

In some embodiments where the surface is a surface of a membrane, themembrane can be configured to separate a first chamber (e.g., a firstmicrochannel) and a second chamber (e.g., a second microchannel) in amicrofluidic device.

In some embodiments, the microfluidic device can be configured tocomprise an organ-on-chip device. An exemplary organ-on-chip cancomprise a first chamber (e.g., a first microchannel), a second chamber(e.g., a second microchannel), and a membrane separating the firstchamber and the second chamber. In these embodiments, a first surface ofthe membrane facing the first chamber can comprise the fixed cellmonolayer (e.g., fixed endothelial cell monolayer) thereon, and a secondsurface of the membrane facing the second chamber can comprisetissue-specific cells adhered thereon. In some embodiments, the membranecan be replaced or embedded with extracellular matrix proteins (e.g.,but not limited to collagen, laminin, etc.). In some embodiments, themembrane can also comprise smooth muscle cells and/or fibroblasts.

In some embodiments, the fixed cell monolayer (e.g., fixed endothelialcell monolayer) can be derived from fixing a layer of cells of the firsttype (e.g., an endothelial cell monolayer) that has been grown on thesurface for a period of time. For example, the layer of cells of thefirst type (e.g., an endothelial cell monolayer) can grow on the surfaceuntil it reaches confluence and is then subjected to a fixationtreatment as described herein.

Various methods for fixing cells that are adhered to a surface are knownin the art and can be used herein to generate a fixed cell monolayer. Insome embodiments, the cell monolayer (e.g., endothelial cell monolayer)can be physically fixed by drying and/or dehydration. In someembodiments, the cell monolayer (e.g., endothelial cell monolayer) canbe physically fixed by exposing to air, and/or washing with alcohol,acetone or a solvent that removes water and/or lipids. In someembodiments, the cell monolayer (e.g., endothelial cell monolayer) canbe fixed with a chemical fixative. Non-limiting examples of chemicalfixatives include formaldehyde, paraformaldehyde, formalin,glutaraldehyde, mercuric chloride-based fixatives (e.g., Helly andZenker's solution), precipitating fixatives (e.g., ethanol, methanol,and acetone), dimethyl suberimidate (DMS), Bouin's fixative, and acombination of two or more thereof. In one embodiment, the chemicalfixative for fixing the cell monolayer (e.g., endothelial cellmonolayer) can comprise paraformaldehyde. In some embodiments, the cellmonolayer (e.g., endothelial cell monolayer) can be fixed with adecellularization solvent that stabilizes surface membrane proteinconfiguration and cytoskeleton of a cell. For example, thedecellularization solvent can comprise an aqueous solution comprising adetergent and/or a high pH solution.

The fixed cell monolayer (e.g., fixed endothelial cell monolayer) can bederived from a cell line or cells collected from a subject. In someembodiments, cells collected from a subject can be reprogrammed to formpluripotent stem cells, which are then differentiated into target cellsto generate a fixed cell monolayer.

The fixed cell monolayer (e.g., fixed endothelial cell monolayer) can bederived from cells of any condition. In some embodiments, the fixed cellmonolayer (e.g., fixed endothelial cell monolayer) can be derived fromhealthy cells. In some embodiments, the fixed cell monolayer (e.g.,fixed endothelial cell monolayer) can be derived from diseased cells. Insome embodiments, the diseased cells can be derived from a subject(e.g., a healthy subject or a subject diagnosed with a disease ordisorder of interest). In some embodiments, the diseased cells can begenerated by contacting healthy cells (e.g., healthy endothelial cells)with a condition-inducing agent (e.g., inflammation-inducing agent)prior to the fixation treatment. The condition-inducing agent (e.g.,inflammation-inducing agent) can comprise a physical stimulus, achemical agent, a biological agent, a molecular agent, or a combinationof two or more thereof.

By detecting interaction between cells (e.g., blood cells such asplatelets) in the fluid sample and the fixed cell monolayer (e.g., fixedendothelial cell monolayer), temporal and/or spatial dynamics of thecells in the fluid sample interacting with each other and/or to thefixed cell monolayer can be measured. In some embodiments, the measuredtemporal and/or spatial dynamics of cell interaction measured cancomprise cell adhesion, cell detachment, cell translocation, and cellembolization/aggregation. In some embodiments, the measured temporaland/or spatial dynamics of cell interaction can comprise bindingdynamics of the cells (e.g., blood cells such as platelets) to the fixedcell monolayer (e.g., fixed endothelial cell monolayer), bindingdynamics of the cells (e.g., blood cells such as platelets) to eachother, or a combination thereof.

Depending on cell detection methods, the cells in the fluid sample canbe label-free or labeled, e.g., with a detectable label. An exemplarydetectable label can comprise a fluorescent label.

Any art-recognized cell detection methods can be used to detectinteraction between the cells in the fluid sample and the fixed cellmonolayer. In some embodiments, an imaging-based method can be used. Anexemplary imaging-based method can comprise time-lapse microscopy.

The inventors have showed that the fixed endothelial cell monolayer canbe stored for a period of time without undermining its applicability toplatelet dynamics analysis. Accordingly, in some embodiments, thesurface comprising the fixed cell monolayer (e.g., fixed endothelialcell monolayer) can have been stored for a period of time prior toflowing the fluid sample over the surface. In some embodiments, thefixed cell monolayer (e.g., fixed endothelial cell monolayer) can bestored at a non-freezing temperature. For example, in some embodiments,the fixed cell monolayer (e.g., fixed endothelial cell monolayer) can bestored at room temperature. In some embodiments, the fixed cellmonolayer (e.g., fixed endothelial cell monolayer) can be stored at atemperature of about 4° C. or lower. In some embodiments, the fixed cellmonolayer (e.g., fixed endothelial cell monolayer) can be stored at atemperature of about 4° C.-10° C.

The period of time to store the fixed cell monolayer (e.g., fixedendothelial cell monolayer) can vary with the selected storagetemperature. In some embodiments, the period of time can be at leastabout 1 day or longer. In some embodiments, the period of time can be atleast about 5 days or longer.

The fluid sample can be flowed over the surface comprising the fixedcell monolayer (e.g., fixed endothelial cell monolayer) at apre-determined shear rate or flow rate. For example, the fluid samplecan be flowed over the surface at a flow rate that generates aphysiological or pathological wall shear rate. For example, thephysiological or pathological wall shear rate can range from about 50sec⁻¹ to about 10,000 sec⁻¹.

The fixed cell monolayer (e.g., fixed endothelial cell monolayer) andthe fluid sample can be derived from the same subject or from differentsources.

In some embodiments, the fixed cell monolayer can comprise a fixedendothelial cell monolayer, and the fluid sample cells can compriseblood cells such as platelets. Accordingly, in these embodiments, thesystem can be used to determine spatial dynamics of blood cells such asplatelets in a fluid sample.

The methods and/or systems described herein can provide tools todiagnose a disease or disorder induced by cell dysfunction or abnormalcell-cell interaction in a subject. Accordingly, another aspectdescribed herein relates to a method of determining if a subject is atrisk, or has, a disease or disorder induced by cell dysfunction orabnormal cell-cell interaction. The method comprises: (a) flowing afluid sample of the subject over a surface comprising a fixed cellmonolayer thereon; (b) detecting interaction of cells in the fluidsample between each other and/or with the fixed cell monolayer; and (d)identifying the subject to be at risk, or have the disease or disorderinduced by cell dysfunction when the cell-cell interaction is higherthan a control; or identifying the subject to be less likely to have adisease or disorder induced by cell dysfunction when the cell-cellinteraction is no more than the control.

In some embodiments, the living or fixed cell monolayer used in themethods described herein can be subject-specific.

In some embodiments, the method of determining if a subject is at risk,or has a disease or disorder induced by cell dysfunction and/or abnormalcell-cell interaction can be used for diagnosis and/or prognosis of adisease or disorder induced by blood cell dysfunction (e.g., plateletdysfunction), and/or guiding and/or monitoring of an anti-plateletand/or anti-inflammation therapy. Accordingly, in some embodiments, thefixed endothelial cell monolayer can comprise a fixed endothelial cellmonolayer. The fixed endothelial cell monolayer can be subject-specific.In some embodiments, the fluid sample can comprise blood cells such asplatelets. Thus, a method of determining if a subject is at risk, or hasa disease or disorder induced by blood cell dysfunction (e.g., plateletdysfunction) is also described herein. Non-limiting examples of thedisease or disorder induced by blood cell dysfunction (e.g., plateletdysfunction) include, but are not limited to thrombosis, an inflammatoryvascular disease (e.g., sepsis, or rheumatoid arthritis), acardiovascular disorder (e.g., acute coronary syndromes, stroke, ordiabetes mellitus), vasculopathies (e.g., malaria, disseminatedintravascular coagulation), or a combination of two or more thereof.

Compositions for determining cell-cell interaction are also describedherein. In one aspect, the composition comprises (a) a solid substratehaving a surface comprising a fixed monolayer of cells of a first typethereon; and (b) a fluid sample in contact with the surface, wherein thefluid sample comprises cells of a second type.

In some embodiments, the fixed monolayer of cells of the first type cancomprise a fixed endothelial cell monolayer. In some embodiments, thecells of the second type in the fluid sample can comprise blood cellssuch as platelets.

In some embodiments, the fluid sample can comprise a blood sample.

The fixed cell monolayer can comprise fixed cells (e.g., fixedendothelial cells), fixed cell extract(s) (e.g., fixed endothelial cellextract(s)), and/or fixed cell-associated proteins (e.g., fixedendothelial cell-associated proteins) that are adhered to the surface.

In some embodiments, the fixed cell monolayer (e.g., fixed endothelialcell monolayer) can be derived from fixing a cell layer (e.g., anendothelial cell monolayer) that has been grown on the surface for aperiod of time, e.g., until the cell layer reaches confluence.

The surface with which the fluid sample is in contact can be a surfaceof any fluid-flowing conduit disposed in a solid substrate. The solidsubstrate can be any solid substrate that is compatible to the fluidsample and the fixed cell monolayer. Non-limiting examples of the solidsubstrate include a cell culture device, a microscopic slide, a cellculture dish, a microfluidic device, a microwell, and any combinationsthereof.

In one embodiment, the surface can be a wall surface of a microchannel.In one embodiment, the surface can be a surface of a membrane. In someembodiments where the surface is a surface of a membrane, the membranecan be configured to separate a first chamber (e.g., a firstmicrochannel) and a second chamber (e.g., a second microchannel) in amicrofluidic device.

In some embodiments, the microfluidic device can be configured tocomprise an organ-on-chip device. An exemplary organ-on-chip cancomprise a first chamber (e.g., a first microchannel), a second chamber(e.g., a second microchannel), and a membrane separating the firstchamber and the second chamber. In these embodiments, a first surface ofthe membrane facing the first chamber can comprise the fixed cellmonolayer (e.g., fixed endothelial cell monolayer) thereon, and a secondsurface of the membrane facing the second chamber can comprisetissue-specific cells adhered thereon. In some embodiments, the membranecan be replaced or embedded with extracellular matrix proteins (e.g.,but not limited to collagen, laminin, etc.). In some embodiments, themembrane can also comprise smooth muscle cells and/or fibroblasts.

For example, in some embodiments, the methods, systems, and/orcompositions described herein can be configured to permit a bloodcell-comprising fluid sample (e.g., platelet-comprising fluid sample)flowing over a more reliable and physiologically relevantendothelialized surface inflamed by a cytokine, thus mimicking the invivo endothelium-blood cell (e.g., platelet) crosstalk environment,e.g., in a normal or diseased state. The blood cell (e.g., platelet)dynamics (e.g., adhesion, translocation and/or detachment) can berecorded and quantified, which is not possible with the existing goldstandard tests. As the blood cell (e.g., platelet) function/interactioncan be reproduced even when the live endothelial cells are fixed, thecompositions with a fixed endothelial cell monolayer described hereincan be stored under standard laboratory conditions for a period of time(e.g., days or weeks) and still remain functional. Thus, thecompositions described herein can be operated near patients' bedside,e.g., in clinics or hospitals, to determine blood cell (e.g., platelet)dysfunction, e.g., for diagnosis of a disease or disorder induced byblood cell (e.g., platelet) dysfunction.

In some embodiments, the compositions described herein can furthercomprise tissue-specific cells. For example, in some embodiments, amicrofluidic device can comprise a first chamber (e.g., a firstmicrochannel), a second chamber (e.g., a second microchannel), and amembrane separating the first chamber and the second chamber, wherein afirst surface of the membrane facing the first chamber can comprise afixed endothelial cell monolayer thereon, and a second surface of themembrane facing the second chamber can comprise tissue-specific cellsadhered thereon. A fluid comprising blood cells (e.g., blood or bloodsubstitute) can be introduced into the first chamber such that bloodcells can interact with the fixed endothelial cell monolayer. In someembodiments, the fixed endothelial monolayer can be an inflamed ordiseased endothelial cell monolayer. By incorporating luminal blood cellfluid transport (e.g., a fluid comprising blood cells such as platelets)over a fixed endothelial cell monolayer and live culture of tissuespecific cells, a physiologically relevant in vitro model of bloodcell-induced inflammation can be created to probe its pathophysiologyand/or to permit drug screening.

Additional aspects of the invention will be apparent to those ofordinary skill in the art in view of the detailed description of variousembodiments, which is made with reference to the drawings, a briefdescription of which is provided below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective illustration of a microfluidic device.

FIG. 2 is top view illustration of a single microfluidic chip with aplurality of microfluidic devices.

FIG. 3 is a fluorescence micrograph of a microchannel covered with humanumbilical vein endothelial cells (“HUVECs”).

FIG. 4A is a confocal immunofluorescence microscopic image showing a topview of a microchannel section with HUVECs.

FIG. 4B shows a front view of the microchannel section of FIG. 4A.

FIG. 4C shows a side view of the microchannel section of FIG. 4A.

FIG. 5 is a graph that shows fluorescence measured after immunostaininga fixed endothelium with ICAM-1.

FIG. 6 is a graph that shows fluorescence measured after immunostaininga fixed endothelium with VCAM-1.

FIG. 7 is a graph that shows fluorescence measured after immunostaininga fixed endothelium with VWF.

FIG. 8 is a graph that shows fluorescence measured after immunostaininga fixed endothelium with a tissue factor.

FIG. 9 is a plurality of representative maximum intensity projectionmicrographs with fluorescently labeled platelets adhering to achemopreserved endothelium.

FIG. 10 is a graph that shows platelet coverage when blood is perfusedinside a microchannel that is lined with a living or fixed endothelium.

FIG. 11A is a fluorescent micrograph showing fibrin that is formed alongwith platelet aggregates on a fixed endothelium (scale bar—200 μm).

FIG. 11B is a fluorescent micrograph showing fibrin that is formed alongwith platelet aggregates on a fixed endothelium (scale bar—20 μm).

FIG. 12 is a graph illustrating platelet coverage on a fixed endotheliumthat is pretreated with TNF-α when blood samples are perfused through amicrofluidic device.

FIG. 13 is a graph illustrating light transmission aggregometry of bloodsamples containing different doses of abciximab using either ADP orcollagen as an agonist.

FIG. 14 is a graph illustrating platelet coverage when blood samplescontaining different doses of the drug abciximab are perfused throughcollage-coated microfluidic devices.

FIG. 15 is a graph illustrating platelet coverage on a fixed endotheliumthat has been pretreated with TNF-α when blood samples from healthydonors are perfused through microfluidic devices.

FIG. 16 is a graph illustrating light transmission aggregometry ofhealthy versus antiplatelet treated blood samples using ADP or collagenas an agonist.

FIG. 17 is a graph illustrating platelet coverage when healthy versussubject blood samples are perfused through collagen-coated microfluidicdevices.

FIG. 18 is an illustration of a microfluidic blood vessel with culturedendothelial cells.

FIG. 19 shows fluorescence micrographs depicting a section of an imagedmicrochannel showing platelet accumulation (left to right) on collagen,a healthy blood vessel, and a TNF-α stimulated vessel.

FIG. 20 shows fluorescence micrographs depicting a section of an imagedmicrochannel with platelet accumulation after 4 minutes of laser-inducedinjury on a mouse cremaster arteriole (scale bar—μm 25).

FIG. 21 shows fluorescent micrographs of a large section of a vascularchamber with intravascular thrombus formation in collagen (top image),and TNF-α stimulated endothelium in a dose dependent manner (bottomthree images).

FIG. 22 is a graph illustrating ICAM-1 expression on the endothelialcells after stimulation with TNF-α.

FIG. 23 is a graph illustrating a sensitivity analysis of a plateletendothelial dynamics algorithm.

FIG. 24 is a conceptual schematic of a human lung showing alveoliinteracting with neighboring blood vessels during hemostasis orpulmonary dysfunction.

FIG. 25 is a perspective view illustration of a microfluidic device withtwo compartments separated by a thin porous membrane.

FIG. 26 is a side view illustration of the microfluidic device of FIG.25.

FIG. 27 shows visual stacks of confocal micrographs with junctionalstructures, after twelve days of co-culture.

FIG. 28 is a chart showing vascular ICAM-1 measured after TNF-αstimulation relative to untreated cells in the presence of alveolarepithelial cells (AE).

FIG. 29 is a chart showing platelet-endothelial dynamics in amicrofluidic device that follows a similar trend as ICAM-1 of FIG. 28.

FIG. 30 shows fluorescent micrographs with platelets (left), fibrin(middle), and merged (right) on an endothelial surface when stimulatedby TNF-α.

FIG. 31 is a chart showing vascular ICAM-1 that is measured after LPSstimulation relative to untreated cells in the presence or absence ofalveolar epithelial cells (AE).

FIG. 32 is a chart showing platelet-endothelial dynamics measured in amicrofluidic device, in the presence or absence of the alveolarepithelial cells (AE).

FIG. 33 shows fluorescence micrographs with platelet aggregates andfibrin at the end of blood perfusion through a microfluidic device.

FIG. 34 is a chart showing barrier permeability measured after LPSstimulation, relative to untreated cells in the presence or absence ofthe alveolar epithelial cells (AE).

FIG. 35 shows representative confocal micrographs with gap junctionsunder no treatment or LPS treatment, in the presence of a blood vesselalone or with epithelium (AE).

FIG. 36 shows fluorescent micrographs illustrating evolution of bloodclots (left to right) in a cremaster artery of the mouse.

FIG. 37 is a chart showing platelet-endothelial dynamics computed onfluorescent time-series of platelets.

FIG. 38 is an illustration showing a microfluidic device that containsalveolar epithelial cells (AE) treated with LPS and a vessel treatedwith parmodulin (PM2).

FIG. 39 is a chart showing platelet-endothelial dynamics that aremeasured in a microfluidic device containing AE cells.

FIG. 40 is a chart illustrating platelet coverage on a microchip coveredwith collagen.

FIG. 41 shows a coefficient of variation (CV) colormap of a singlethrombus formed in a laser injured mouse in vivo.

FIG. 42 shows histological sections representing sections of a mouselung with clots.

FIG. 43 shows a representative kymograph of a small section of a channelwith an attachment and detachment pattern of platelets on a collagensurface.

FIG. 44 shows a representative kymograph of a small section of a channelwith an attachment and detachment pattern of platelets on a vessel thatis untreated.

FIG. 45 shows a representative kymograph of a small section of a channelwith an attachment and detachment pattern of platelets on a vessel thatis TNF-α treated.

FIG. 46 shows a chart shows a coefficient of variance (CV) of afluorescence signal observed over time at a representative single pixellocation of an image time-series of platelet accumulation, as plotted inthe kymographs shown in FIGS. 43-45.

FIG. 47 shows a top image representative of a coefficient of variation(CV) colormap of a large section of a vessel, and a bottom image with agraph showing the CV across the length of the channel at arepresentative width location for a collagen-treated vessel.

FIG. 48 shows a top image representative of a coefficient of variation(CV) colormap of a large section of a vessel, and a bottom image with agraph showing the CV across the length of the channel at arepresentative width location for an untreated vessel.

FIG. 49 shows a top image representative of a coefficient of variation(CV) colormap of a large section of a vessel, and a bottom image with agraph showing the CV across the length of the channel at arepresentative width location for vessel treated with TNF-α.

FIG. 50 shows a graph illustrating the interpercentile range(95^(th)-5^(th) percentile value) of the coefficient of variation (CV)plotted in the graphs illustrated in FIGS. 47-49, as a measure ofdepicting spatial heterogeneity in platelet accumulation.

FIG. 51 illustrates an exemplary organ-on-chip (OOC) device inaccordance with one embodiment of the present disclosure.

FIG. 52 is a cross-section of the organ-on-chip (OOC) device taken alongline 52-52 of FIG. 51, illustrating first and second microchannels ofthe organ-on-chip (OOC) device.

FIG. 53 is a cross-section of the organ-on-chip (OOC) device taken alongline 53-53 of FIG. 52, illustrating fluid flow between the firstmicrochannel and the second microchannel of the organ-on-chip (OOC)device of FIG. 51.

FIGS. 54A-54E are images and schematic diagrams of a biomimetic plateletfunction analyzer (μPFA) according to one or more embodiments describedherein. (FIG. 54A) Schematic of the multilayered microfluidic devicecomprising a first channel and a second channel, wherein the firstchannel and the second channel are separated by a permeable membrane.The side of the membrane facing the first channel can comprise anendothelium adhered thereto. The other side of the membrane facing thesecond channel can comprise astrocytes or other cell types of interest.Shear stresses and/or fluid flow (e.g., whole blood or blood flow) canbe induced in the first channel. The multilayered microfluidic devicecan optionally comprise a vacuum channel on one or both sides of thefirst and the second channel. (FIG. 54B) Picture of one embodiment of adevice showing blood passing through it when pulled by a syringe pump.(FIG. 54C) Schematic drawing (top view) of the vascular chamber showinginlet, outlet and optional pressure ports. (FIG. 54D) A fluorescentmicrograph of endothelial cells (green/top channel, CD31 staining) andastrocytes (red/bottom channel/GFAP staining) co-cultured in oneembodiment of the device described herein. Such device can then beperfused with blood. (FIG. 54E) (left panels) A sectional view of thevascular chamber coated with HUVECs and inflamed with tumor necrosisfactor (TNF-α). (right panels) The endothelial ICAM-1 expression isincreased with increase in TNF concentration.

FIG. 55 is a set of data showing platelet adhesion on differentsurfaces. (Left panel) Bar graph showing area-averaged platelet adhesionrate on collagen, unstimulated endothelium and cytokine-stimulatedendothelium. (Right panels) Snapshots of a section of the vascularchamber after 15 minutes of whole blood flow containing labeledplatelets. Scale bar=50 μm. ** P<0.001

FIG. 56 is a set of data showing temporal dynamics of plateletsinteracting with different surfaces. (Left panel) Bar graph showingTemporal Platelet Dynamics (TPD) indices varying with differentsurfaces, namely, collagen, unstimulated endothelium andcytokine-stimulated endothelium. (Right panels) Kymographs of a sectionof the vascular chamber perfused with whole blood containing labeledplatelets. Scale bar=50 μm (vertical direction). ** P<0.001

FIG. 57 is a set of data showing spatial dynamics of plateletsinteracting with different surfaces. (Left panel) Bar graph showingSpatial Platelet Dynamics (SPD) indices varying with different surfaces,namely, collagen, unstimulated endothelium and cytokine-stimulatedendothelium. (Right panels) Time-averaged coefficient of variation (CV)maps of a section of the vascular chamber perfused with whole bloodcontaining labeled platelets. Scale bar=50 μm. ** P<0.001

FIG. 58 is a set of micrographs showing formaldehyde-fixed humanumbilical vein endothelial cells (HUVECs) in the device according to oneor more embodiments described herein. (Left panel) von Willebrand factorstaining (green). (Right panel) Tissue factor (TF) staining (green).

FIGS. 59A-59C are bar graphs showing area averaged platelet adhesion(FIG. 59A), Temporal Platelet Dynamics (TPD) (FIG. 59B) and SpatialPlatelet Dynamics (SPD) (FIG. 59C) of platelets over collagen (COL*) andendothelium fixed for 1 day or 5 days (ENDO*). ** P<0.001

FIG. 60 is a set of confocal images showing a cross-section of atwo-compartment organ-on-a-chip with co-cultures of HUVECs (topcompartment) and human astrocytes (bottom compartment). The HUVECs andastrocytes were both treated overnight with 100 ng/ml TNF-α. Theendothelial compartment (in which the endothelial cells were cultured onall walls of a channel) was perfused with whole blood for about 15minutes. (Left panel) Platelets (red) were observed to be mainly on thewalls of the endothelial compartment, while fibrin has formed mostly inthe static (no shear) astrocyte compartment due to the reaction betweenblood fibrinogen and thrombin. The fibrin passed through the endothelialcompartment (high shear). (Right panel) F-actin/nuclear staining showsastrocyte localizations on the membrane and the floor of the bottomcompartment.

FIG. 61 is a block diagram showing an exemplary system for use in themethods described herein, e.g., for determining temporal and/or spatialdynamics of cells (e.g., platelets) binding to each other and/or a cellmonolayer (e.g., an endothelial cell monolayer).

FIG. 62 is a block diagram showing an exemplary system for use in themethods described herein, e.g., for determining temporal and/or spatialdynamics of cells (e.g., platelets) binding to each other and/or a cellmonolayer (e.g., an endothelial cell monolayer).

FIG. 63 is an exemplary set of instructions on a computer readablestorage medium for use with the systems described herein to determinetemporal dynamics of cells (e.g., platelets) binding to each otherand/or a cell monolayer (e.g., an endothelial cell monolayer).

FIG. 64 is an exemplary set of instructions on a computer readablestorage medium for use with the systems described herein to determinespatial dynamics of cells (e.g., platelets) binding to each other and/ora cell monolayer (e.g., an endothelial cell monolayer). In someembodiments, the exemplary set of instructions can further comprise aportion of the instructions from FIG. 63 to compute temporal dynamics ofthe cells (e.g., platelets) binding to each other and/or a cellmonolayer. When both Aggregation (spatial) Index (FIG. 64) andEmbolization (temporal) Index (FIG. 63) are used to determine celldynamic behavior, to diagnose disease, and/or to monitor therapy, insome embodiments, both indices can be greater than their respectivecontrol values. In some embodiments, both indices can be lower thantheir respective control values. In some embodiments, one index can begreater than its respective control value, while another index can belower than its respective control value. For example, samples frompatients with hypercoagulable disorders can show normal/strong plateletaggregation (e.g., represented by a high Aggregation Index), but verylow “embolization.”

FIGS. 65A-65C depict an embodiment of a platelet dynamics assessmentdevice as described herein. FIG. 65A depicts a schematic of themicrofluidic device for quantifying platelet dynamics on a livingendothelium under flow when cultured within a hollow microchannel (400μm wide, 100 μm high, 2 cm long). Human whole blood is stored in areservoir at the inlet (left) and pulled by a syringe pump attached tothe outlet (right) at a flow rate of 30 μl/min (shear rate: 750 sec⁻¹).Fluorescently tagged platelets that interact with the endothelium arevisualized over time within a central region of the long section of thechannel using automated microscopy. FIG. 65B depicts a photograph of themicrofluidic platelet assessment chip containing 6-channels (bar, 15mm). FIG. 65C depicts a representative fluorescence micrograph ofplatelet-rich thrombi that form on the TNF-α treated endothelial surfacein this device when whole blood is perfused. The thrombi contain bothplatelets (red) and fibrin (green) (bar, left: 100 μm, right: 25 μm). A3-dimensional confocal reconstruction of platelet-rich thrombi formed onthe endothelium-lined microfluidic channel, stimulated by cytokine TNF-αcan be generated.

FIGS. 66A-66B depict the comparison of platelet aggregation on collagenversus living endothelium. FIG. 66A depicts the image acquisition andanalysis protocol according to one embodiment described herein.Fluorescent micrographs were acquired every 30 sec for a total of 15min; 10 (1×10) image tiles were captured at each time step and stitchedtogether to form a panoramic view, resulting in an image time series(K). FIG. 66B depicts fluorescence micrographs of the microchannel whencoated with collagen (COL; top) or lined with endothelium (HUVEC;bottom) are shown on the left. Representative image tiles of plateletsinteracting with the collagen-coated surface (top) or the surface ofendothelium stimulated with different doses of TNF-□ (bottom) 10 minafter initiating blood flow are shown at the right (bar, 200 μm).

FIGS. 67A-67B depict the quantitative analysis of platelet adhesion andthrombus formation using an Aggregation Index (AI). FIG. 67A depictsrepresentative coefficient of variance (CV) maps, produced using the“fire” color map, showing platelet adhesion patterns on a collagensurface (COL) versus endothelial (HUVEC) lined surface stimulated withdifferent doses of TNFα. Color bar indicates the intensity ofaggregation/thrombi (white is greatest; blood flow was from left toright; bar, 200 μm). FIG. 67B depicts a graph showing plateletaggregation indices (AI) derived from maps shown in FIG. 67A. The timeseries stack (K) is projected across time computing the temporalcoefficient of variance (CV) at each spatial pixel (M), and the AI isthe inter-quartile range (IQR) of M. Note that the unstimulatedendothelium does not induce platelet adhesion or thrombus formation,whereas the amount and variability of the aggregation pattern increasesin TNFα dose-dependent manner on stimulated endothelial cells; thisresults in a rise of AI with increasing TNFα dose (0 ng TNFα/ml; 5 ngTNFα/ml; 100 ng TNFα/ml; n=3,**p<0.01).

FIGS. 68A-68B depict the quantitative analysis of translocation andembolization of platelet-rich thrombi using an Embolization Index (EI).FIG. 68A depict representative size-adjusted kymographs showingembolization pattern of platelets on a collagen surface (COL) orendothelium (HUVEC) stimulated with different TNFα doses. FIG. 68Bdepicts a graph showing platelet Embolization Indices (EI) derived fromkymographs shown in FIG. 68A. The time series stack (K) was averagedacross the width of the channel and a kymograph (a temporal map ofplatelet dynamics) was generated (N); the EI is the CV of N. Note that,platelets remained adherent to the collagen surface over time and didnot translocate, resulting in a low EI. The unstimulated endothelium didnot induce translocation and/or embolization of platelet-rich thrombiwhereas these dynamical processes increased in a TNFα dose-dependentmanner (0 ng TNFα/ml; 5 ng TNFα/ml; 100 ng TNFα/ml; n=3,**p<0.01).

FIGS. 69A-69E depict whole blood platelet analysis on chemicallypreserved endothelium in a microchannel according to one embodimentdescribed herein. FIG. 69A is a schematic diagram depicting plateletthrombus formation over a natural versus chemopreserved endothelium. Ina microchannel covered on all sides with an untreated livingendothelium, whole blood flows without clotting (left). In contrast,platelet-rich thrombus forms if the endothelium is prestimulated by aproinflammatory cytokine, such as TNF-α, due to expression ofprocoagulatory proteins at its surface (right). The responses of bloodunder flow shown in the figures at the left can be reconstituted usingsimilar microchannels that are lined by a chemically preservedendothelium. FIG. 69B depicts a schematic of one embodiment of themicrodevice described herein. The inlet is a blood reservoir (dia. 3.5mm) and it is pulled via a syringe pump at the outlet (dia. 1.5 mm)connected to tubing (not shown). The dotted region (˜2.5 mm×500 μm) isvisualized over time using automated fluorescence microscopy. FIG. 69Cdepicts endothelial engineering on the microchip. Confocalimmunofluorescence microscopic images show a section of the microchannelcontaining adherent HUVECs when viewed from above (left) and in sideview (right). The dotted region represents the analyzed area of plateletaccumulation (green, VE cadherin; blue, nuclear DAPI; bar, 200 μm). FIG.69D depicts quantitative analysis of platelet adhesion and aggregationon living vs. chemopreserved endothelial substrate. (Left) The plateletcoverage on both living (white bar) and fixed (shaded bar) endotheliumincreases in a TNF-α dose-dependent manner (n=4). No significantdifference in platelet coverage was observed using living versuschemopreserved endothelium. (Right) Representative maximum intensityprojection micrographs showing fluorescently labeled platelet-richthrombi adhering to the chemopreserved endothelium in a TNF-αdose-dependent manner. The statistical analysis was performed using2-way ANOVA (Sidak's multiple comparison test). (bar, 200 μm). FIG. 69Edepicts Tissue Factor (TF, blue) and von Willebrand Factor (vWF, purple)expression on untreated (white bar) vs. stimulated (shaded bar)chemopreserved endothelial substrate. Error bars, standard error of mean(s.e.m.). * P<0.05 in all graphs.

FIGS. 70A-70F are bar graphs depicting analysis of the chemopreservedendothelium covered microchip to monitor antiplatelet therapy. FIG. 70Ashows that the platelet coverage over the TNF-α stimulatedchemopreserved endothelium decreases with increase in abciximab drugconcentration (n=4). The statistical analysis was performed using 1-wayANOVA (Sidak's multiple comparison test). FIG. 70B shows that when bloodis perfused at a shear of 750 sec⁻¹ on a collagen microfluidic device,there is an insignificant decrease in platelet adhesion and aggregationwith increase in abciximab dosage (n=3) whereas, (as shown in FIG. 70C)platelets aggregate in the presence of ADP (white bar) and collagen(shaded bar) as agonists, only when control blood is used. In thepresence of drug, no aggregation is observed on the light transmissionaggregometry (n=5). (For FIGS. 70B and 70C, N.S=non-significant;statistical analysis based on 1-way ANOVA (Sidak's multiple comparisontest)). FIG. 70D shows that an untreated chemopreserved endothelium(white bar) is quiescent for both healthy donors and patients who are onchronic use of aspirin alone or both aspirin and clopidogrel, but incomparison to healthy donors, patients result in significantly lowerplatelet coverage on the TNF-α stimulated chemopreserved endothelium(shaded bar)(n=11). The statistical analysis was performed using one-wayANOVA (Sidak's multiple comparison test). FIG. 70E shows that blood fromsubjects who are on antiplatelet therapy, showed insignificantdifference in aggregation compared to healthy controls using a collagencoated microfluidic device (n=11, p=0.4493—non-significant based onunpaired t-test results (two-tailed)). FIG. 70F shows that blood fromsubjects who are on antiplatelet therapy, exhibited significantly lessaggregation compared to healthy controls using a light transmissionaggregometry with ADP (white bar) and collagen (shaded bar) as agonists.(n=11, *: P<0.05 based on 2-way ANOVA analysis (Sidak's multiplecomparison test)). * P<0.05 in all graphs. N.S.=non-significant.

FIGS. 71A-71B depict the engineering of one embodiment of a responsiveendothelium-lined microfluidic channel described herein. FIG. 71Adepicts confocal immunofluorescence microscopic images showing theentire length of the microchannel containing adherent human umbilicalcord endothelial cells (HUVECs) shown when viewed from above (Top) andin cross-sectional views (Bottom) (green, VE cadherin; blue, nuclearDAPI; bar, 300 μm). To generate a 3-dimensional confocal reconstructionof endothelium-lined microfluidic channel, sequential images obtainedalong the microchannel containing adherent human umbilical cordendothelial cells (HUVECs) were acquired using Leica SP5×MP invertedconfocal microscope. The virtual volume was processed using Huygensdeconvolution software and rendered with Imaris (Green, VE cadherin;blue, nuclear DAPI). FIG. 71B depicts a graph (left) andimmunofluorescence microscopic views of the cultured endothelium stainedfor intercellular adhesion molecule-1 (ICAM-1) at left or F-actin atright, showing dose-dependent activation of ICAM-1 (left images) whenstimulated with tumor necrosis factor alpha (TNF-α)(green, ICAM-1 orF-actin; blue, DAPI; bar, 300 μm).

FIG. 72 is a set of bar graphs showing expression of tissue factor (TF)and von Willebrand factor (vWF), respectively, on TNF-α inflamedendothelium. HUVECs were cultured on PDMS-coated 24 well plates for 48 hand left untreated or stimulated with 5 or 100 ng/ml TNF-α. The effectof TNF-α on TF (left panel) and vWF (right panel) expression on theendothelial cell surface was estimated by measuring immunofluorescenceintensity, normalized with respect to the untreated case. (*P<0.05, n=3)

FIGS. 73A and 73B are data graphs showing area averaged plateletadhesion rate. FIG. 73A depicts area averaged platelet adhesion rate ona surface calculated using automated Otsu image thresholding algorithm.The percentage area covered was calculated as the ratio of number ofpixels with the value of unity to the size of the binary image andplotted against time. The dotted line is the linear regression curve fitand the adhesion rate is the slope of the regression line. FIG. 73Bdepicts area-averaged platelet adhesion rate. n=3,**p<0.01

FIG. 74 is a set of data graphs showing platelet adhesion andaggregation dynamics. Graphical representation of the coefficient ofvariance (CV) image M(x,y). On a collagen (COL) and healthy endothelial(HUVEC 0 ng/ml) surface, the range of variance is narrow. However, theplatelet patterns on TNF-α treated endothelium (HUVEC) are heterogeneousand fluctuate in a dose-dependent manner. The inter-quartile range (IQR)of the signal is termed platelet aggregation index (AI).

FIG. 75 shows an exemplary image acquisition and analysis protocol forplatelet coverage. Platelets were visualized using time-lapsefluorescence imaging (LD Plan Neofluar 20×, NA 0.4; Zeiss Axio Observer;Hamamatsu ORCA C11440 CMOS digital camera) using an exposure time of 200ms. Images were tiled to create a composite panoramic view (18,600pixels long and 2,050 pixels wide; 1 pixel=0.325 μm). In step 1, atimeseries (K) of a 10-frame panorama (6 mm long×0.665 mm wide region ofthe microchannel), at a lapse of every 30 seconds was recorded. Imageswere archived as OME-TIFF format files, and image analysis was performedusing Zeiss Zen 2012 imaging software and MATLAB 2014 routines. Theresulting image stack was maximum intensity projected along time (step2), thresholded, segmented (step 3) and cropped to the central 200 μm ofthe channel width (step 4) for analysis. Finally, platelet coverage wascomputed from the binary image as the ratio of bright pixels (intensityvalue=1) to the total number of pixels in the image (step 5).

FIG. 76 is a set of fluorescent images showing platelet-rich thrombusformation in the microfluidic device after blood perfusion. Fluorescentmicrograph shows fibrin (green) is formed along with platelet aggregates(red) in collagen and TNF-α (5 ng/ml) stimulated chemopreservedendothelium after recalcified citrated whole blood is perfused throughthe device. The platelet aggregates are small and more uniformlydistributed over the collagen compared to the inflamed endothelialsurface. (bar, 100 μm)

While the invention is susceptible to various modifications andalternative forms, specific embodiments have been shown by way ofexample in the drawings and will be described in detail herein. Itshould be understood, however, that the invention is not intended to belimited to the particular forms disclosed. Rather, the invention is tocover all modifications, equivalents, and alternatives falling withinthe spirit and scope of the invention as defined by the appended claims.

DETAILED DESCRIPTION

While this invention is susceptible of embodiment in many differentforms, there is shown in the drawings and will herein be described indetail preferred embodiments of the invention with the understandingthat the present disclosure is to be considered as an exemplification ofthe principles of the invention and is not intended to limit the broadaspect of the invention to the embodiments illustrated.

As used herein, the term “monolayer” refers to a single layer of cellson a growth surface, on which no more than 10% (e.g., 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, 1%, or 0%) of the cells are growing on top of oneanother, and at least about 90% or more (e.g., at least about 95%, atleast 98%, at least 99%, and up to 100%) of the cells are growing on thesame growth surface. In some embodiments, all of the cells are growingside-by side, and can be touching each other on the same growth surface.The condition of the cell monolayer can be assessed by any methods knownin the art, e.g., microscopy, and/or immunostaining for cell-celladhesion markers. In some embodiments where the fixed cell monolayercomprises a fixed endothelial cell monolayer, the condition of theendothelial cell monolayer can be assessed by staining for anyart-recognized cell-cell adhesion markers in endothelial cells, e.g.,but not limited to VE-cadherin.

In some embodiments, the monolayer can be substantially confluent. Asused herein, the term “confluent” generally indicates that the cellshave formed a coherent monocellular layer on a growth surface, so thatvirtually all the available growth surface is used. As used herein, theterm “substantially confluent” indicates that most of the cells growingon the same surface are in contact with each other around the cellperiphery, and interstices can remain, such that over about 70% or more,including, e.g., over about 80%, over about 90%, over about 95% or more,and up to 100%, of the available growth surface is used. The term“available growth surface” as used herein refers to sufficient surfacearea to accommodate a cell. Thus, small interstices between adjacentcells that cannot accommodate an additional cell do not constitute“available growth surface.” Accordingly, in some embodiments, the fixedcell monolayer (e.g., fixed endothelial cell monolayer) can be derivedfrom fixing a layer of cells of the first type (e.g., an endothelialcell monolayer) that has been grown on the surface for a period of time.In some embodiments, the period of time can vary with degree ofconfluence, cell proliferation rate, number of initially seeded cells,number of cell culture passages, or a combination thereof. For example,the layer of cells of the first type (e.g., an endothelial cellmonolayer) can grow on the surface until it is substantially confluentand is then subjected to a fixation treatment as described herein. Insome embodiments where the layer of cells of the first type comprisesendothelial cells, the endothelial cell culture can be cultured for atleast about 24 hours or longer to reach an intact and confluentmonolayer.

In some embodiments, the monolayer can be exposed to or stimulated by anagent, e.g., a condition-inducing agent, prior to fixation. In theseembodiments, the cells in the monolayer can overgrow and form plexiformlesions, prior to fixation.

The fixed cell monolayer (e.g., fixed endothelial cell monolayer) can bederived from a cell line (e.g., primary cell lines), stem cells, orcells collected from a subject. In some embodiments, target cells to becultured on the surface can be collected from a subject. For example, toform a fixed endothelial cell monolayer, endothelial cells can becollected from a subject. In some embodiments, endothelial cells can bederived from stem cells or induced pluripotent stem cells. For example,skin fibroblasts can be collected from a subject and reprogrammed toform pluripotent stem cells, which are then differentiated intosubject-specific target cells (e.g., endothelial cells) to form a fixedcell monolayer (e.g., a fixed endothelial cell monolayer). Methods toderive different types of differentiated cells from induced pluripotentstem cells are known in the art. For example, vascular endothelium canbe derived from induced pluripotent stem cells using the methods asdescribed, e.g., in Adams et al., Stem Cell Reports (2013) 1:105-113.

The fixed cell monolayer (e.g., fixed endothelial cell monolayer) can bederived from cells of any condition or state (e.g., but not limited towild-type, healthy state, mutant, disease-specific, and stimulatedstate). In some embodiments, the fixed cell monolayer (e.g., fixedendothelial cell monolayer) can be derived from healthy cells orwild-type cells. As used herein, the term “healthy” refers to a statewithout any symptoms of any diseases or disorders, or not identifiedwith any diseases or disorders, or not on any physical, chemical and/orbiological treatment, or a state that is identified as healthy byskilled practitioners based on microscopic examinations. As used herein,the term “wild-type” refers to a natural state without any geneticmanipulation.

In some embodiments, the fixed cell monolayer (e.g., fixed endothelialcell monolayer) can be derived from disease-specific cells. As usedherein, the term “disease-specific” refers to a state of cells thatrecapitulates at least one characteristic associated with a disease,disorder or an injury, or different stages thereof. In some embodiments,the term “disease-specific” can refer to a specific stage or grade of adisease, disorder or an injury. Examples of diseases, disorders, orinjuries can be related to any organ or tissue, e.g., but not limitedto, blood vessel, lung, brain, nerve network, blood-brain-barrier,vascular, kidney, liver, heart, spleen, pancreas, ovary, testis,prostate, skin, eye, ear, skeletal muscle, colon, intestine, andesophagus. In some embodiments where the fixed cell monolayer comprisesa fixed endothelial cell monolayer, the endothelial cells canrecapitulate at least one characteristic associated with a vascularand/or inflammatory disease or disorder.

The disease-specific cells can be either obtained from a biopsy of apatient carrying the disease, disorder or an injury, or inducing ahealthy cell with a condition-inducing agent that is known to induce thecell to acquire at least one characteristic associated with the disease,disorder, or injury, prior to a fixation treatment. For example, acondition-inducing agent can comprise an environmental or physical agentsuch as radiation; a chemical or biological agent, e.g., but not limitedto, cytokines described herein and/or pathogens; a molecular agent(e.g., but not limited to a pathogen-derived toxin such aslipopolysaccharides (LPS), and/or a candidate drug/compound that isknown to cause endothelial activation or thrombotic toxicity), or acombination of two or more thereof.

In some embodiments, the fixed cell monolayer (e.g., fixed endothelialcell monolayer) can be derived from stimulated cells. As used herein,the term “stimulated” refers to a state of cells that are responsive toa condition-inducing agent exposed to them. As used herein, the term“condition-inducing agent” refers to any agent that can cause a cell todisplay a phenotype that is deviated from a basal state (withoutexposure to the condition-inducing agent). The condition-inducing agentcan provoke a beneficial or adverse effect such as cytotoxic effect onthe cells. Examples of a condition-inducing agent can include, but arenot limited to, environmental or physical agents such as radiation(e.g., gamma radiation) and mechanical stress (e.g., fluid shearstress); proteins, peptides, nucleic acids, antigens, cytokines, growthfactors, toxins, cells (including prokaryotic and eukaryotic cells suchas virus, bacteria, fungus, parasites, and mammalian cells),particulates (e.g., smoke particles or asbestos), particles (e.g.,nanoparticles or microparticles, magnetic particles), small molecules,biologics, and any combinations thereof.

In some embodiments where the fixed cell monolayer comprises a fixedendothelial cell monolayer, the condition-inducing agent added to theendothelial cell monolayer prior to fixation can comprise aninflammation-inducing agent that induces endothelium inflammation and/oractivation. The inflammation-inducing agent can comprise one or acombination of two or more of the following: physical conditions (e.g.,lack of oxygen, and disturbed flow patterns), chemical (e.g., glucoselevels, environmental pollutants), biochemical (e.g., inflammatorymolecules such as interleukins, interferons, TNF-superfamily molecules),biological, human cell derived (complex mixtures), or biological,non-human cell derived (e.g., bacteria, or factors secreted bybacteria). In some embodiments, the inflammation-inducing agent cancomprise at least one or more (e.g., at least two or more)proinflammatory cytokines such as IL-6, IL-2, IL-10, sCD40L,interleukins and interferons, which can be applied to produceendothelial activation and inflammation that are involved in plateletfunction, activation and aggregation. Other factors such aslipopolysaccharide (LPS), toxins (Shiga toxin etc.), bacteria, viruses,nanoparticles, antibodies and drug candidates can also be used asinflammation-inducing agents to stimulate the endothelium. In someembodiments, the inflammation-inducing agent can comprise glucose. Forexample, the surface comprising the fixed endothelial cell monolayer canbe configured to provide highly fluctuating levels of glucose tostimulate a “diabetic” surface. In some embodiments, complex mixtures ofsoluble factors from tumor cells, and/or activated white blood cells canbe used as the inflammation-inducing agents to activate the endothelium.In some embodiments, the inflammation-inducing agents can comprisetemporary co-culture with tumor cells and/or white blood cells to induceendothelial activation.

As used herein, the term “fixed,” “fixation,” or “fixing” means thatcell-associated components or materials, including, e.g., but notlimited to whole cells, cell fragments, intracellular proteins,extracellular proteins (e.g., secreted proteins, cell surfacereceptors), nucleic acid molecules, and/or cytoskeleton, are treatedwith a fixative agent or composition, resulting in at least a partialstabilization or preservation of their molecular position, histologicalstructure, and/or molecular function. Upon fixation, whole cells are notalive anymore but proteins and/or nucleic acid of the cells andcell-associated proteins and/or nucleic acid present in the cellmonolayer remain stable and functional (e.g., ability to induce aresponse in other live cells). Thus, cell fixation can provide spatialheterogeneity and expressions of proteins and/or nucleic acid moleculesthat would be expected in live culture and/or in vivo. In someembodiments, a fixation agent or composition can result in at leastabout 50% or more (including, e.g., at least about 60%, at least about70%, at least about 80%, at least about 90%, at least about 95%, atleast about 98%, or more or up to 100%) stabilization or preservation ofthe cell-associated components' or materials' molecular position,histological structure, and/or molecular function.

Various methods for fixing cells that are adhered to a surface are knownin the art and can be used herein to generate a fixed cell monolayer. Insome embodiments, cell fixation methods can be selected to keep wholecell intact. For example, in some embodiments, the cell monolayer (e.g.,endothelial cell monolayer) can be physically fixed by drying and/ordehydration. For example, in some embodiments, the cell monolayer (e.g.,endothelial cell monolayer) can be physically fixed by exposing the cellmonolayer to air, and/or washing the cell monolayer with a dryingsolvent, e.g., alcohol (e.g., ethanol) and acetone, or a solvent thatremoves water and/or lipids. In some embodiments, the cell monolayer(e.g., endothelial cell monolayer) can be fixed with a chemicalfixative. Non-limiting examples of chemical fixatives includeformaldehyde, paraformaldehyde, formalin, glutaraldehyde, mercuricchloride-based fixatives (e.g., Helly and Zenker's solution),precipitating fixatives (e.g., ethanol, methanol, and acetone), dimethylsuberimidate (DMS), Bouin's fixative, and a combination of two or morethereof. In one embodiment, the chemical fixative for fixing the cellmonolayer (e.g., endothelial cell monolayer) can compriseparaformaldehyde. Accordingly, in some embodiments, the fixed cellmonolayer (e.g., endothelial cell monolayer) can be derived by fixingwhole cells adhered on a surface.

In some embodiments, the cell layer (e.g., endothelial cell monolayer)can be fixed with a fixative agent or composition comprisingparaformaldehyde.

Additionally or alternatively, cell fixation methods can be selected toremove a portion of cell components and fix the remaining cellcomponents. For example, in some embodiments, the cell monolayer (e.g.,endothelial cell monolayer) can be fixed with a decellularizationsolvent. The decellularization solvent is a solvent that partially orcompletely removes or extracts cell membranes and/or soluble componentsbut stabilizes molecular configuration, molecular function, andmolecular position of surface membrane proteins (e.g., membrane proteinreceptors), insoluble components, and/or cytoskeleton of a cell. Methodsfor fixing cells by membrane removal or extraction are known in the art,e.g., as described in Ben-Ze'ev et al., Cell (1979) 17: 859-865; Pouratiet al., Am J Physiol. (1998) 274: C1283-1289; Sims et al., J Cell Sci.(1992) 103: 1215-1222; and Fey et al., J Cell Biol. (1984) 98:1973-1984. F or example, in some embodiments, the decellularizationsolvent can comprise an aqueous solution comprising a detergent (e.g.,polysorbate surfactants such as Tween 20, and/or a nonionic surfactantsuch as Triton X-100; glycosides such as saponins) and/or a high pHsolution (e.g., an alkaline solution such as ammonium hydroxide).Accordingly, in some embodiments, the fixed cell monolayer can bederived from fixing cell extract and/or cell-associated proteins thatare adhered to the surface.

In some embodiments, an additive can be added to a fixative agent orcomposition to render cells permeable to ligands which bind tointracellular moieties. Binding of ligands (e.g., but not limited to,antibodies and detectable labels) to intracellular moieties can bedesired for purposes of visualizing, detecting, or isolating the cellsafter they have been preserved. Additives that increase the permeabilityof cell membrane and/or nuclear envelopes are known in the art. Forexample, the organic solvent acetone, methanol, and/or ethanol canincrease the permeability of cell membrane when preservation of proteinand/or nucleic acid moieties is/are desired.

In some embodiments, an additive can be added to a fixative agent orcomposition to keep cells isosmotic which helps preservation. Forexample, sugars such as sucrose and/or buffered solutions can be addedto keep cells isosmotic.

A skilled artisan can optimize the concentrations of a fixative agent orcomposition added to the cell monolayer (e.g., an endothelial cellmonolayer). Typical concentrations of the fixative agent or compositioncan range from about 1% (v/v) to about 10% (v/v), and they can vary withthe strength of the selected fixative agent or composition. For example,when paraformaldehyde is used to fix the cell monolayer (e.g.,endothelial cell monolayer), the concentration of the paraformaldehydecan range from about 1% (v/v) to about 8% (v/v). In one embodiment, thecell monolayer (e.g., endothelial cell monolayer) can be fixed withparaformaldehyde at a concentration of about 4%.

The temperature at which the cells are fixed can range from about 0° toabout room temperature. In some embodiments, the fixation temperaturecan vary from about 0° C. to about 10° C. In some embodiments, thefixation temperature can vary from about 0° C. to about 4° C. In oneembodiment, the fixation temperature can be about 4° C.

The fixation time duration (i.e., time elapsing before the cell layer(e.g., endothelial cell monolayer) is fixed once a fixative agent orcomposition is added) can vary with a number of factors, including,e.g., but not limited to types, temperature and/or concentration of theselected fixative agent or composition. For example, the higher theconcentration of a fixative agent or composition is used, the shorterthe fixation time duration can be. For example, when paraformaldehyde ata concentration of about 4% is used to fix the cell monolayer (e.g., anendothelial cell monolayer), the fixation time duration can range fromabout 15 minutes to about 30 minutes. In one embodiment, the fixationtime duration can be about 20 minutes.

The inventors have showed that once the cell monolayer (e.g.,endothelial cell monolayer) is fixed, the fixed cell monolayer (e.g.,endothelial cell monolayer) can be stored for a period of time withoutsignificantly reducing its applicability, e.g., to determine plateletdynamics analysis, as compared to a freshly fixed cell monolayer or afixed cell monolayer that has been stored for a shorter period of time.Accordingly, in some embodiments, the surface comprising the fixed cellmonolayer (e.g., fixed endothelial cell monolayer) can have been storedfor a period of time prior use. In some embodiments, the fixed cellmonolayer (e.g., fixed endothelial cell monolayer) can be stored at anon-freezing temperature. For example, in some embodiments, the fixedcell monolayer (e.g., fixed endothelial cell monolayer) can be stored atroom temperature. In some embodiments, the fixed cell monolayer (e.g.,fixed endothelial cell monolayer) can be stored at a temperature ofabout 4° C. or lower. In some embodiments, the fixed cell monolayer(e.g., fixed endothelial cell monolayer) can be stored at a temperatureof about 4° C.-10° C.

The period of time to store the fixed cell monolayer (e.g., fixedendothelial cell monolayer) without significantly reducing itsapplicability (i.e., shelf-life of the surface comprising a fixed cellmonolayer (e.g., a fixed endothelial cell monolayer)) can vary with theselected storage temperature. In some embodiments, the period of time(shelf-life) can be at least about 1 day or longer, including, e.g., atleast about 2 days, at least about 3 days, at least about 4 days, atleast about 5 days or longer. In some embodiments, the period of time(shelf-life) can be at least about 5 days or longer. In someembodiments, the period of time (shelf-life) can be at least about 1week or longer. In some embodiments, the period of time (shelf-life) canbe at least about 2 weeks or longer, including, e.g., at least about 3weeks, at least about 4 weeks or longer.

In some embodiments where the fixed cell monolayer comprises a fixedendothelial cell monolayer, the fluid sample can comprise a bloodsample, a serum sample, a plasma sample, a lipid solution, a nutrientmedium, or a combination of two or more thereof. In some embodiments,the fluid sample can comprise at least one type of blood cells, e.g.,red blood cells, white blood cells, and platelets. In one embodiment,the fluid sample can comprise platelets, and no red blood cells. In thisembodiment, the method described herein can further comprise removingred blood cells from the fluid sample (e.g., a blood sample) prior toflowing the blood sample over the surface.

Without wishing to be bound by theory, platelet function may depend uponthe presence of Ca²⁺ and Mg²⁺. Thus, for a fluid sample comprising acitrated blood sample (where citration of a blood sample generallyquenches the free Ca²⁺ and Mg²⁺ ions to prevent blood coagulation),addition of Ca²⁺ (e.g., calcium chloride) and Mg²⁺ (magnesium chloride)to the fluid sample can help restore the native physiological state ofthe platelet, e.g., to allow platelet aggregation or coagulation. Thus,in some embodiments, the citrated blood sample can be added with Ca²⁺(e.g., calcium chloride) and Mg²⁺ (magnesium chloride) such that thefinal concentrations reach about 4-12 mM and 3-10 mM, respectively.However, when a blood sample is collected in the presence of thrombinblockers to prevent blood coagulation (e.g., but not limited to heparin,hirudin, EDTA, PPACK and/or any other anticoagulant), the addition ofCa²⁺ (e.g., calcium chloride) and Mg²⁺ (magnesium chloride) may not berequired.

In some embodiments, the surface can be a surface of a fluid-flowingconduit or passageway disposed in a solid substrate. In someembodiments, the solid substrate can comprise a cell or tissue culturedevice, including, e.g., but not limited to a transwell, a microwell, amicrofluidic device, a bioreactor, a culture plate, or any combinationsthereof.

In some embodiments, the surface can be a solid surface. For example, inone embodiment, the solid surface can be a wall surface of a fluidchannel, e.g., a microfluidic channel.

In some embodiments, the surface can be a porous or gas-permeablesurface. For example, in one embodiment, the surface can be a surface ofa gas-permeable membrane. In some embodiments, the membrane can beconfigured to separate a first chamber (e.g., a channel or acompartment) and a second chamber (e.g., a channel or a compartment) ina cell or tissue culture device.

In some embodiments, the surface can be disposed in a microfluidicdevice. In one embodiment, the microfluidic device can be anorgan-on-a-chip device. Examples of various organ-on-a-chip devices,e.g., as described in International Patent Application Nos: WO2010/009307, WO 2012/118799, WO 2013/086486, WO 2013/086502, and in U.S.Pat. No. 8,647,861, the contents of each of which are incorporatedherein by reference in their entireties, can be utilized to perform themethods described herein. In one embodiment, the organ-on-a-chip devicecan comprise a first channel and a second channel separated by amembrane. The membrane can be porous (e.g., permeable or selectivelypermeable), non-porous (e.g., non-permeable), rigid, flexible, elastic,or any combination thereof. In some embodiments, the membrane can beporous, e.g., allowing exchange/transport of fluids (e.g., gas and/orliquids), passage of molecules such as nutrients, cytokines and/orchemokines, cell transmigration, or any combinations thereof. In someembodiments, the membrane can be non-porous. In some embodiments, afirst surface of the membrane facing the first channel comprises a fixedcell monolayer (e.g., a fixed endothelial cell monolayer) adheredthereon. In some embodiments, a second surface of the membrane facingthe second channel can comprise tissue-specific cells adhered thereon.In some embodiments, the membrane can be replaced or embedded withextracellular matrix proteins (e.g., but not limited to collagen,laminin, etc.). In some embodiments, the membrane can also comprisesmooth muscle cells and/or fibroblasts.

By detecting interaction between cells (e.g., blood cells such asplatelets) in the fluid sample and the fixed cell monolayer (e.g., fixedendothelial cell monolayer), temporal and/or spatial dynamics of thecells in the fluid sample interacting with each other and/or to thefixed cell monolayer can be measured. In some embodiments, the measuredtemporal and/or spatial dynamics of cell interaction measured cancomprise cell adhesion, cell detachment, cell translocation, and cellembolization/aggregation. As used herein, the term “cell adhesion”refers to spatial and/or temporal adhesion of cells (e.g., platelets) toeach other and/or to a fixed cell monolayer (e.g., fixed endothelialcell monolayer) when the fluid sample flows over the fixed cellmonolayer (e.g., fixed endothelial cell monolayer). As used herein, theterm “cell detachment” refers to spatial and/or temporal detachment ofcells from cell-cell binding (e.g., between platelets) and/or from thefixed cell monolayer (e.g., fixed endothelial cell monolayer) when thefluid sample flows over the fixed cell monolayer (e.g., fixedendothelial cell monolayer). As used herein, the term “celltranslocation” refers to temporal movement of cells (e.g., platelets)from one position to another when the fluid sample flows over the fixedcell monolayer (e.g., fixed endothelial cell monolayer). As used herein,the term “cell embolization/aggregation” refers to spatial and/ortemporal binding of cells (e.g., platelets) to form an aggregate, clump,or embolic material when the fluid sample flows over the fixed cellmonolayer (e.g., fixed endothelial cell monolayer).

In some embodiments, the measured temporal and/or spatial dynamics ofcell interaction can comprise binding dynamics of the cells (e.g., bloodcells such as platelets) to the fixed cell monolayer (e.g., fixedendothelial cell monolayer), binding dynamics of the cells (e.g., bloodcells such as platelets) to each other, or a combination thereof.

Depending on cell detection methods, the cells (e.g., blood cells suchas platelets) in the fluid sample can be label-free or labeled. In someembodiments, the cells (e.g., blood cells such as platelets) can belabel-free. In these embodiments, phase-contrast or brightfieldmicroscopy can be used to detect the cells when they are flowing acrossthe surface comprising a fixed cell monolayer. In some embodiments wherethe methods described herein are used for platelet function analysis,the label-free platelets can be detected by phase-contrast orbrightfield microscopy. In some embodiments where red blood cells mayobscure the view, the fluid sample can be pre-treated to remove redblood cells, or formation of platelet aggregates can be analyzed by anindirect method, e.g., assessment of red blood cell streamlines around agrowing platelet aggregate. Methods for analyzing formation of anaggregate in a label-free manner are known in the art, including, forexample, but not limited to microscopy and local impedance spectroscopy(a physical, electrophysiological measurement).

In some embodiments, the cells (e.g., blood cells such as platelets) canbe labeled. As used herein, the term “labeled” refers to a cell beingmanipulated to express or carry a detectable label, e.g., to facilitatedetection of the presence or absence of the cell. As used herein, theterm “detectable label” refers to a composition capable of producing adetectable signal indicative of the presence of a target. Detectablelabels suitable for the detection methods that provide spatial and/ortemperate information about cell dynamics (e.g., cell adhesion, celldetachment, cell translocation, and/or cell embolization/aggregation)described herein can include any composition detectable byspectroscopic, photochemical, biochemical, immunochemical, electrical,magnetic, optical, chemical means, or a combination of two or morethereof. In some embodiments, the detectable label can encompass anyimaging agent (e.g., but not limited to, a fluorophore, a nanoparticle,and/or a quantum dot).

An exemplary detectable label can comprise a fluorescent label orfluorophore. A wide variety of fluorescent reporter dyes are known inthe art. Typically, the fluorophore is an aromatic or heteroaromaticcompound and can be a pyrene, anthracene, naphthalene, acridine,stilbene, indole, benzindole, oxazole, thiazole, benzothiazole, cyanine,carbocyanine, salicylate, anthranilate, coumarin, fluorescein, rhodamineor other like compound.

In some embodiments, the detectable label can be conjugated to acell-targeting moiety. For example, platelets can be labeled with adetectable label that is conjugated to a platelet-targeting moiety.Examples of platelet-targeting moieties include, but are not limited to,platelet endothelial cell adhesion molecule (e.g., CD31), antibodies toplatelet surface protein (e.g., CD41, CD61, and/or CD42b).

Any art-recognized cell detection methods can be used to detectinteraction between cells in the fluid sample and the fixed cellmonolayer. In some embodiments, an imaging-based method can be used. Anexemplary imaging-based method can comprise time-lapse microscopy,wide-field holography, stereomicroscopes, cameras, compact mobiledevices, or any combinations thereof.

The fluid sample can be flowed over the surface comprising a fixed cellmonolayer (e.g., fixed endothelial cell monolayer) at a pre-determinedshear rate or flow rate. For example, the fluid sample can be flowedover the surface at a flow rate that generates a physiological orpathological wall shear rate. In some embodiments, the fluid sample canbe flowed over the surface at a flow rate that generates a physiologicalor pathological arterial shear rate. For example, the physiological orpathological wall shear rate can range from about 50 sec⁻¹ to about10,000 sec⁻¹. In some embodiments, the physiological or pathologicalshear rate can range from about 100 sec⁻¹ to about 1,000 sec⁻¹. In someembodiments, the physiological or pathological shear rate can range fromabout 200 sec⁻¹ to about 900 sec⁻¹. In one embodiment, the physiologicalor pathological shear rate can be about 750 sec⁻¹. Various methods toflow a fluid sample over a surface in a chamber are known in the art.For example, the fluid transport over the surface comprising a fixedcell monolayer (e.g., fixed endothelial cell monolayer) can be achievedby syringe pump, capillary driven flow, gravitational flow and/orpressure-driven flow.

The fixed cell monolayer (e.g., fixed endothelial cell monolayer) andthe fluid sample can be derived from the same subject or from differentsources.

As used herein, the term “reactive fluid sample cells” refers to cellsfrom a fluid sample having a higher frequency (e.g., by at least about30% or more) of binding with each other (e.g., aggregation), and/or witha fixed cell monolayer (e.g., adhesion, detachment, and translocation),as compared to control cells (e.g., healthy cells, or nonstimulatedcells).

As used herein, the terms “treat” or “treatment” or “treating” refers toboth therapeutic treatment and prophylactic or preventative measures,wherein the object is to prevent or slow the development of the disease,such as slow down the development of a blood cell-induced disease ordisorder, or reducing at least one effect or symptom of the bloodcell-induced disease or disorder. Treatment is generally “effective” ifone or more symptoms or clinical markers are reduced as that term isdefined herein. Alternatively, treatment is “effective” if theprogression of a disease is reduced or halted. That is, “treatment”includes not just the improvement of symptoms or markers, but also acessation of at least slowing of progress or worsening of symptoms thatwould be expected in absence of treatment. Beneficial or desiredclinical results include, but are not limited to, alleviation of one ormore symptom(s), diminishment of extent of disease, stabilized (i.e.,not worsening) state of disease, delay or slowing of diseaseprogression, amelioration or palliation of the disease state, andremission (whether partial or total), whether detectable orundetectable. “Treatment” can also mean prolonging survival as comparedto expected survival if not receiving treatment.

As used herein, the term “platelet dysfunction” refers to abnormalplatelet behavior, as compared to healthy platelets. In one embodiment,platelet dysfunction can be caused by increased adhesion to anendothelium (e.g., by at least about 30% or more), as compared tohealthy platelets. In one embodiment, platelet dysfunction can be causedby abnormal detachment from other platelets and/or from an endothelium(e.g., by at least about 30% or more), as compared to healthy platelets.In one embodiment, platelet dysfunction can be caused by abnormaltranslocation (e.g., by at least about 30% or more), as compared tohealthy platelets. As used herein, the term “abnormal translocation”refers to a platelet that gets activated in one location and deposits atanother location to form a clot or cause inflammation response. Forexample, thromboembolism can be considered as abnormal translocation. Inone embodiment, platelet dysfunction can be caused by increasedaggregation between platelets (e.g., by at least about 30% or more), ascompared to healthy platelets.

Generally, in reference to one aspect, a simple microfluidic device islined by a chemically preserved human endothelium that retains itsability to support thrombus formation and platelet adhesion as bloodflows through its channels at an arterial shear rate. This biomimeticdevice demonstrates the potential practical value for laboratory andpoint-of-care settings by showing that it can be more rapid and reliablethan standard aggregometry or similar collagen-coated microfluidicdevices.

Generally, in reference to another aspect, a lung-on-a-chip microfluidicdevice reconstitutes critical functional aspects of intravascularpulmonary thrombus formation and platelet-endothelial dynamics in thevicinity of an endotoxin or cytokine stimulated primary human alveolarepithelium. The lung-on-a-chip microfluidic device is perfused withhuman whole blood in a bioengineered vascular lumen. Additionally, basedon inherent dynamic complexity and spatiotemporal heterogeneity in theway platelets interact with the vessel wall and initiate thrombusformation, a method is directed to a large-scale real-time fluorescenceimage acquisition and a statistical algorithm for quantifyingplatelet-endothelial dynamics during endothelial dysfunction.

Combining the supporting quantitative analysis and the in vitroorgan-level functional microfluidic device, the pulmonarythrombosis-on-chip model has been extended to evaluate thecytoprotective affect of a potential protease activator receptor(“PAR-1”) inhibitor, termed parmodulin 2 (“PM2”). Thus, the results leadto the proposition that this inhibitor is a potential anti-thromboticand anti-inflammatory therapeutic drug for human patients to treatdisorders that lead to acute lung thrombosis.

I. Fixed Cells—Materials and Methods

A. Microfluidic Device Design and Treatment

By way of example, a microfluidic device is fabricated usingphotolithography followed by soft lithography with polydimethysiloxane(“PDMS”). According to this example, six microfluidic devices are placedon a single PDMS mold of the size of a standard glass slide, e.g., 75×25millimeters (“mm”). The microfluidic devices are first exposed to oxygenplasma for 30 seconds using, for example, a PE-100 benchtop plasmasystem from Plasma Etch. Then, the microfluidic devices are treated with1% (3-aminopropyl)-trimethoxysilane (“APTMS”), from Sigma, in 100%anhydrous ethanol for ten minutes. After subsequent rinsing with 70% and100% ethanol, the microfluidic devices are dried and type I collagenfrom rat tail, e.g., 100 micrograms/milliliter (“μg/ml) from Corning, isintroduced in microchannels of the microfluidic devices.

The microfluidic devices are left overnight at 37° C. in a 5% carbondioxide (“CO₂”) incubator, after which they are rinsed with EndothelialGrowth Medium-2 (“EGM-2”) from Lonza. Human Umbilical Vein EndothelialCells (“HUVEC”) from a mixed donor, by Lonza, are kept in culture andsuspended at 12.5 million cells/milliliter (“mL”) in EGM-2, afterconfluence. The suspension is introduced into the collagen-coatedmicrochannels, after which the microfluidic devices are incubated upsidedown for 20 minutes. A fresh HUVEC suspension is then introduced in themicrochannels, after which the microfluidic devices are incubated foreight hours to promote cell attachment across the channel. Themicrochannels are, then, rinsed with EGM-2, sometimes containing afreshly prepared solution of tumor necrosis factor (“TNF-α”), which is arecombinant from E. coli from Sigma. Antibodies against intercellularadhesion molecule-1 (“ICAM-1”) and vascular adhesion molecule-1(“VCAM-1”), tissue factor (from Santa Cruz), VWF (from Abcam) andVE-Cadherin (from Santa Cruz) were perfused into the microfluidicdevices after fixing the endothelium with 2% paraformaldehyde for tenminutes, incubating the endothelium for three hours, and counterstainingwith a secondary fluorescent IgG antibody for three hours.

B. Blood Samples and Human Subjects

Human blood (e.g., received from Research Blood Components, Cambridge,Mass.) is acquired in 3.2% sodium citrate tubes and is used within 5hours of blood draw, to prevent pre-analytical effects on plateletfunction. Institutional review board (“IRB”) approval is obtained foruse of discarded blood samples. Subjects are selected from amongpatients who re taking antiplatelet medication. A total of 11 samplesare used for analysis, from which 8 patients re on aspirin alone and 3re on aspirin and clopidogrel (Plavix).

C. Blood Perfusion

500 microliters (“μL”) of whole blood are pipetted into a fluidreservoir fitted to one end of a microchannel. Platelets are labeledwith human CD41-PE antibody (e.g., 10 μL/mL from Invitrogen) that isdirectly added to the blood samples and incubated at room temperaturefor 10 minutes. Fluorescently labeled fibrinogen (e.g., 10 μg/mL fromLife Sciences) is added, if required. Blood is pulled through the device(e.g., 30 μL/min) via tubing at an outlet using a syringe pump (e.g.,PHD Ultra™ CP, Harvard Apparatus), resulting in an arterial shear rateof 750 s⁻¹. After two minutes, blood is supplemented with 100millimolars (“mM”) calcium (“CaCl₂”) and 75 mM magnesium (“MgCl₂”) tosupport coagulation-activated blood clotting (e.g., 100 μL/mL).

D. Image Acquisition and Analysis

Platelets are visualized using time-lapse fluorescence imaging (e.g.,20×, NA 0.4). A time series of a 10-frame panorama (e.g., 6 mmlong×0.665 mm wide region of a microchannel), at a lapse of every 30seconds is recorded. The resulting image stack is maximum intensityprojected along time, thresholded, segmented, and cropped to a central200 microns (“μm”) of the channel width for analysis. Finally, plateletcoverage is computed from the binary image as the ratio of bright pixelsto the total number of pixels in the image.

E. Light Transmission Aggregometry (“LTA”)

The LTA is performed in accordance with manufacturer instructions. Forexample, the LTA uses adenosine diphosphate (“ADP”) 10 μM and collagen 2μg/mL from Chrono-Log.

F. Statistical Analysis

All data is presented as mean±standard error (SEM). Two-tailed P valuesare obtained from a statistical t-test or one way ANOVA using GraphPadPrism V6.

II. Fixed Cells—Results and Discussion

A. Formation and Evaluation of a Chemically Preserved Endothelium

In reference to FIG. 1 and the above example, a microfluidic device 100is engineered that contains a rectangular microchannel 102. By way ofexample, the microchannel 102 is 400 μm wide, 100 μm high, and 2centimeters (“cm”) long. The microfluidic device 100 has a blood inletreservoir 104, followed by the straight microchannel 102 that ends at anoutlet 106. A syringe pump is attached to the outlet 106 to pull theblood from the microchannel 102.

In reference to FIG. 2, a microfluidic device 200 is made of PDMS andhas six similar and independent microchannels 202 that are provided on asingle chip. The microchannels 202 are similar to or identical with themicrochannel 102 illustrated in FIG. 1. According to one example, thechip 200 is 15 cm long and is fabricated on a glass slide.

In reference to FIGS. 3 and 4A-4C, the inner surface of the rectangularmicrochannel 102 is coated with collagen and, then, is cultured withHUVECs to create a tube lined by a continuous, confluent endothelialcell monolayer. Specifically, FIG. 3 illustrates a fluorescencemicrograph that shows the entire microchannel covered with HUVECsstained with VE-cadherin junction marker (scale bar—1 mm). FIGS. 4A-4Cillustrate confocal immunofluorescence microscopic images showing asection of the microchannel 102 with HUVECs when viewed from above inthe xy plane (FIG. 4A), and reconstruction of cross-sectional views fromthe front yz plane (FIG. 4B) and the side xz plane (FIG. 4C). Thisdemonstrates full coverage of all microfluidic microchannel walls. Inthis example, the microfluidic device 100 includes VE-Cadherin, nuclearDAPI, with a scale bar of 200 μm.

Multiple endothelial adhesion molecules are involved in the recruitmentof blood cells and platelets in thrombosis in vivo. In previous studies,treatment of a living endothelium cultured in microfluidic channels withTNF-α results in an increase in expression of surface adhesionmolecules, such as ICAM-1 and VCAM-1 within four hours after addition.To explore whether a fixed endothelium retains expression of these andother surface molecules that could potentially exacerbate thrombosis,the endothelium is first activated in the device by adding increasingdoses of TNF-α, e.g., 0, 5, and 100 nanograms/millliter (“ng/ml”), forapproximately 18 hours. The endothelium cells are fixed with 4%paraformaldehyde in phosphate-buffered saline (“PBS”) for 15 minutes atroom temperature, are rinsed three times with PBS, and, then, stored inPBS at 4° C. in a humid environment.

In reference to FIGS. 5-8, graphs show fluorescence (normalized by theuntreated endothelium) measured after immunostaining the fixedendothelium, which continues to exhibit a dose-dependent increase inICAM-1 (FIG. 5), VCAM-1 (FIG. 6), VWF (see. 7), or tissue factor (FIG.8). In the graphs, *P<0.05 versus untreated and n=3. These resultsindicate that the fixed endothelium retains expression of multiplemolecules that mediate adhesion of blood cells and platelets afteractivation with TNF-α, and induce clotting.

B. Fibrin and Platelet Function Analysis Using a Fixed Endothelium

To explore whether the fixed endothelium in the microfluidic devicesretains the ability to promote hemostasis, recalcified citrated wholeblood (coagulation activated) is immediately perfused through amicrofluidic channel lined by the fixed endothelium and preserved for24-36 hours. Platelet adhesion is analyzed for 15 minutes of flow.

Referring generally to FIGS. 9-11B, platelet coverage and fibrinformation is illustrated on a fixed endothelium in the microfluidicdevice 100. When blood from a healthy donor is flowed over anendothelium that is fixed without prior treatment with TNF-α, there isvirtually no platelet adhesion on the surface, as would be expected fora healthy endothelium. In contrast, when microfluidic devices are usedwith endothelium that is treated with increasing doses of TNF-α prior tofixation, a dose-dependent increase in platelet surface adhesion to theendothelial layer is observed.

Referring more specifically to FIG. 9, representative maximum intensityprojection micrographs show fluorescently labeled platelets adhering toa chemopreserved endothelium in a TNF-α does-dependent manner (scalebar—100 μm). Referring to FIG. 10, a graph shows platelet coverage whenblood is perfused inside the microchannel 102 that is lined with aliving or fixed endothelium, which has been stimulated by TNF-α beforefixation. Of note, no significant difference is observed in plateletadhesion when comparing living versus chemopreserved endothelium, withor without treatment with TNF-α. For example, P>0.05 at each TNF-αconcentration (n=4, *P<0.05). Referring to FIGS. 11A and 11B,fluorescent micrographs show fibrin that is formed along with plateletaggregates on a fixed endothelium, which has been pretreated with TNF-α(5 ng/ml) and perfused with recalcified citrated whole blood (FIG.11A—scale bar—200 μm; FIG. 11B—scale bar—20 μm).

These results confirm that the fixed surface of the endothelium retainsits pro-thrombotic function after fixation. Furthermore, when perfusingwhole blood containing fluorescently labeled fibrinogen, the thrombialso contains a significant amount of fibrin if the endothelium waspre-treated with TNF-α before fixation. This further confirms that thepreserved endothelial surface also retains its ability to activate thecoagulation cascade. The morphology of these thrombi also appear similarto that of thrombi formed on living endothelium in vivo, andsignificantly different from bare collagen-coated flow. Together, theseresults demonstrate that the fixed endothelium is capable of reproducingphysiologically-relevant thrombus formation in our microfluidic device.

C. Potential Clinical Utility of the Device Lined with Fixed Endothelium

In accordance with another exemplary embodiment, a microfluidic devicecontaining a fixed endothelium is used to detect antiplatelet drugeffects in healthy donors and patients taking antiplatelet medication.For example, the cultured endothelium is pretreated with aphysiologically relevant dose of TNF-α (e.g., 5 ng/mL). The microfluidicdevice contains the fixed activated endothelium with whole blood from ahealthy donor containing 0 to 100 μg/mL (e.g., a clinical range ˜1-10μg/mL) of the antiplatelet GP IIb/IIIa antagonist, abciximab (ReoPro®).

Referring to FIG. 12, a graph illustrates platelet coverage on a fixedendothelium that is pretreated with TNF-α when blood samples, whichcontain different doses of the drug abciximab, are perfused through amicrofluidic device (such as the microfluidic device 100 illustrated inFIG. 1). Thus, when the microfluidic device is perfused, adose-dependent inhibition of platelet adhesion is observed, with optimaleffects observed at 10 μg/mL or higher. This is consistent with previousstudies using flow cytometric analysis.

Referring to FIG. 13, a graph illustrates light transmissionaggregometry of blood samples containing different doses of abciximabusing either ADP or collagen as an agonist (n=4). Thus, in contrast tothe results shown in FIG. 12, all concentrations of the drug abciximabproduce virtually complete inhibition of platelet aggregation (e.g., nodose dependence) as detected using LTA, regardless of whether ADP orcollagen was used as an agonist.

Referring to FIG. 14, a graph illustrates platelet coverage when bloodsamples containing different doses of the drug abciximab are perfusedthrough collage-coated microfluidic devices (n=4). While there appearsto be a small suppressive effect on platelet adhesion when the sameblood samples are flowed through an acellular collagen-coated flowchamber, the sensitivity is extremely low and the differences betweenabciximab doses are not statistically significant.

Thus, the microfluidic device containing the fixed endothelium providesan optimally sensitive measure of platelet function, with a higherdynamic response across a range of abciximab concentrations thanexisting platelet function assays. These results suggest that a fixedendothelialized microfluidic device is likely useful in monitoringantiplatelet regimens in patients and has functional advantages overacellular conventional assays. These results also indicate that thefixed surface of the endothelium retains its ability to modulateplatelet interactions via a GPIIb/IIIa pathway, the target of abciximab,which is involved in multiple thrombotic and vascular processes.

Referring to FIG. 15, a graph illustrates platelet coverage on a fixedendothelium that has been pretreated with TNF-α when blood samples fromhealthy donors, versus subjects treated with antiplatelet drugs, areperfused through microfluidic devices (n=11). Thus, whole blood isperfused from subjects who are regular users of antiplatelet drugs, withthe subjects showing a significant reduction in platelet aggregationwhen tested using the microfluidic devices, in comparison to healthydonors.

Referring to FIG. 16, a graph illustrates light transmissionaggregometry of healthy versus antiplatelet treated blood samples usingADP or collagen as an agonist (n=11). Thus, while similar results areobtained using conventional LTA, a microfluidic assay in accordance withthe present disclosure requires a significantly reduced time period tocomplete (e.g., only 15 minutes) relative to the much longer time periodfor an aggregometry test (which must account for sample preparationtime).

Referring to FIG. 17, a graph illustrates platelet coverage when healthyversus subject blood samples are perfused through collagen-coatedmicrofluidic devices (n=11; *P<0.05). Thus, platelet inhibition in thesesubjects is not reliably detected on a collagen-coated flow chamber asthere is no significant difference in platelet coverage between normalcontrols and subjects. Accordingly, the embodiments described abovedemonstrate that a microfluidic device containing fixed endothelium ispotentially applicable in point-of-care settings.

One benefit of a microfluidic device, which contains human endothelialcells that are chemically preserved by fixation, is that it can bestored, shipped, and used when required, either in a laboratory settingor in point-of-care settings. Another benefit of such a microfluidicdevice is that it is a functional assay used to evaluate plateletaggregation and inhibition with drugs in blood samples of patients. Yetanother benefit of such a microfluidic device is that it provides anassay with increased sensitivity than existing assays, and is helpful inrapid analysis of platelet function and hemostasis while incorporatingcontributions from the endothelium and dynamic blood flow.

Although the fixed endothelium may lose some of its live in vivofunctions (e.g., release of bioactive messengers like nitric oxide), andthe exact mechanism by which the surface promotes platelet aggregationand thrombosis is most likely multi-factorial, the above-discussedresults suggest that for a period of 15 minutes of blood flow, the fixedendothelium retains its ability to prevent blood clotting underunstimulated conditions. The results further suggest that the fixedendothelium promotes platelet aggregation and thrombosis when pretreatedwith TNF-α prior to fixation. Notably, the qualitative and quantitativepro-thrombotic and pro-coagulant responses of the fixed endotheliumclosely mimic those of the living endothelium, suggesting that the fixedendothelium also permits the study of thrombus formation on a surfacethat mimics an inflamed endothelium, such as might be found in anatherosclerotic plaque.

III. Algorithm—Results

A. Morphological and Quantitative Analysis of Thrombus Formation

Referring to FIG. 18, a microfluidic device 300 in the form of abioengineered microfluidic blood vessel contains cultured endothelialcells 302 on all walls of a microchannel 304 (also referred to as avascular chamber). According to one embodiment, the microfluidic device300 is similar to or identical with any of the microfluidic devices 100,200 described above. The vessel 300 and endothelial cells 302 areintended to mimic morphology of a blood clot seen in vivo. Thus, wholeblood (containing fluorescently labelled platelets) is perfused throughthe microfluidic device 300.

The morphology of thrombus and platelet-endothelial dynamics, whichoccurs in the microfluidic device 300, is characterized via an imagingand quantitative analysis technique. Specifically, the techniquecharacterizes the morphology as it may occur in vivo and as a result ofendothelial cells activation, blood cells, and shear stress. Forexample, imaging of recalcified citrated whole blood, which containslabelled platelets perfused inside a small section of a vascular chamber304 (no epithelial cell culture), platelet adhesion on a bare collagensurface occurs rapidly, firmly, and increasing steadily over a timespanin the range of about 10 minutes (e.g., 2.5-12.5 minutes).

Referring to FIG. 19, fluorescence micrographs depict a section of theimaged microchannel 304 showing platelet accumulation (left to right) oncollagen, a healthy blood vessel, and a TNF-α stimulated vessel. Thisimaging is consistent with past observations and is reminiscent offormation of a hemostatic plug under vascular injury. In contrast, whenthe lumen chamber 304 is covered with a continuous living endothelialmonolayer over collagen, very little platelet interactions and aggregateformation occur over the course of an experiment, much as what isobserved in blood flowing in a healthy human blood vessel. However, whenendothelial cells are stimulated with an inflammatory cytokine tumornecrosis factor (e.g., TNF-α; 100 ng ml⁻¹) prior to blood flow, plateletadhesion again occurs. Nevertheless, the morphology of the thrombus isclearly distinct from aggregates formed on the collagen surface.

Referring to FIG. 20, fluorescence micrographs depict a section of theimaged microchannel 304 showing platelet accumulation after 4 minutes oflaser-induced injury on a mouse cremaster arteriole (scale bar—μm 25).The typical size of aggregates on activated endothelium is visiblylarger and, interestingly, the size, shape, and organization of thethrombi formed on the activated endothelium in this in vitro modelcorrelates well with what is observed in a mouse model of laser-inducedthrombosis in vivo. This confirms the patency of the in vitro setup inreproducing physiologically-relevant thrombus formation, which ismissing in simple collagen-coated devices.

Referring to FIG. 21, fluorescent micrographs of a large section of thevascular chamber 304 shows intravascular thrombus formation in collagen(top image), and TNF-α stimulated endothelium in a dose dependent manner(bottom three images). The scale bar is 100 μm. The micrographs arehelpful in identifying quantitative parameters for a comparative andcumulative analysis of a large number of platelet-endothelialinteractions that occur over a long region in the microfluidic device300.

Specifically, the analysis includes a method to quantitate plateletfunction in flow chambers and microfluidic devices that has beenprimarily limited to analyzing platelet adhesion on bare collagensurfaces. To analyze platelet-endothelial dynamics that will act as arobust readout of physiologically-relevant clotting, an automatedimaging program creates an image time-series, K(x, y, t), containing a10-frame panorama in space. Images are acquired at a frame rate of 2panoramic images per minute, for a total time of 15 minutes. Anon-dimensional stochastic index is derived to quantitate plateletendothelial dynamics (“P-E”), which is the interpercentile range of acoefficient of variance of the image time-series:

P−E=range(CV(K(x,y,t)  (1)

A feature of this analytical readout is that instead of an “ensembleaveraging,” the method incorporates the cell-surface interactions at thepixel level and quantitates statistical “dispersion” of interactions.

Referring to FIG. 22, a graph illustrates ICAM-1 expression on theendothelial cells after stimulation with TNF-α. Specifically, the graphillustrates testing of the sensitivity of parameter (P-E) in response tochanges in endothelial activation, and the stimulation of theendothelial vessel with various doses of TNF-α, which result in adose-dependent surface expression of adhesion molecule ICAM-1 (n=3).

Referring to FIG. 23, a graph illustrates a sensitivity analysis of theplatelet endothelial dynamics algorithm, showing that in conditions ofhemostasis (e.g., vascular injury/collagen or healthy endothelium), thedynamics are near absent. However, the dynamics increase in a TNF-α dosedependent manner. The platelet endothelial dynamics are also sensitiveto applied shear rate (e.g., n=3, *P<0.05). Thus, a dose-dependenteffect in P-E, shows that the method is sensitive to vasculopathyinduced thrombosis (e.g., n=4).

In fact, unlike platelet adhesion (such as illustrated in FIG. 40), theP-E on a bare collagen surface is extremely low. This shows that thepresent method distinguishes between the conditions of hemostasis (e.g.,in a healthy vessel or vascular injury) and thrombosis due toinflammation. Furthermore, shear stress is a major determinant, forexample, of endothelial function, blood rheology, platelet activation,or immune function, which, together, can alter thrombosis.

The P-E parameter is also sensitive to applied shear stress, and isindicative of a higher tendency to form platelet-rich thrombi andplatelet-endothelial interactions, as shear is increased (see FIG. 22).In contrast, area-averaged platelet adhesion is not sensitive to shear(see FIG. 40). Notably, a CV colormap of a single thrombus formed in alaser injured mouse in vivo, shows high reactivity at the boundarycompared to the central core, which is also observed in individualthrombi that are formed in a microfluidic device, in vitro (see FIG.41).

Interestingly, these regional heterogeneities in the thrombus that arerevealed in accordance with the method described above, have also beenconfirmed by computational studies and also in vivo, showing that athrombus consists of a stable core region surrounded by reactiveunstable shell. Furthermore, the method described above shows that theplatelet-endothelial dynamics (P-E) parameter is a robust parameter thatis applicable to quantitate platelet function and thrombosis, both invitro and in vivo, where the endothelial function is also included.

B. Engineering of the Pulmonary Thrombosis-On-Chip

Referring generally to FIGS. 24-26, to model physiologically-relevantpulmonary hemostasis and thrombosis, a lung-on-a-chip device not onlyallows co-culture of lung epithelial and endothelial cells in thepresence of physiological relevant shear, but also includes primaryhuman cells and a more functional arterial blood vascular lumen in whichhuman whole blood can be perfused. Referring specifically to FIG. 24, aconceptual schematic of a human lung 400 shows that the alveoli 402interacts with neighboring blood vessels 404 during hemostasis orpulmonary dysfunction. Referring specifically to FIGS. 25 and 26, amicrofluidic device 500 contains two PDMS compartments 502 (whichinclude a top compartment 502A and a bottom compartment 502B) separatedby a thin porous membrane 504 that reproduces the microarchitecture ofthe alveolar-capillary interface. The microfluidic device 500, accordingto this example, is in the form of a lung-on-a-chip device. The topcompartment 502A is cultured with human primary alveolar epithelialcells 506 and the entire bottom chamber 502B is lined with humanendothelial cells 508 forming a lumen. Whole blood is perfused throughthe bottom chamber 502B and thrombus formation is visualized usingfluorescence microscopy from the bottom. Optionally, or alternatively,the compartments 502A, 502B are in the form of channels.

Referring to FIG. 27, visual stacks of confocal micrographs showjunctional structures, after twelve days of co-culture, of a singlelayer of the primary alveolar epithelium 506 at the top chamber 502A(stained with e-cadherin) and endothelial monolayers 508 covering eitherside of the lower chamber 502B (stained with ve-cadherin), through whichblood perfusion takes place. The scale bar for the top and bottom imagesof FIG. 27 is 50 μm, and the scale bar for the middle image is 250 μm.

Thus, in the alveolar chamber 502A, human primary alveolar epithelialcells (“AE”) are cultured and in the blood vessel chamber 502B, theentire chamber 502B is cultured with human endothelial cells (μBV) for atotal of 12 days, thus creating an organ-level functional device 500where the epithelial, endothelial, and blood cell interactions arevisualized and analyzed in real-time, in the presence of whole bloodflow and conditions that mimic thrombus formation in vivo. Notably, thislung-on-a-chip device 500 contains co-culture of healthy human primaryalveolar cells and healthy endothelial cells that show barrier integrityand intact junctions even after 12 days of living culture, across theentire length and breadth of the device 500. Accordingly, in the stateof hemostasis (e.g., healthy cell culture and perfusion of healthyblood), perfuse recalcified citrated whole blood (coagulation activated)is perfused for up to 20 minutes without any observed platelet adhesionor clotting inside the lumen of the lung-on-chip device 500. Thisconfirms the formation of a healthy organ-level functional microfluidicdevice 500 that is capable of resembling the state of hemostasis.

Referring to FIG. 28, a chart shows vascular ICAM-1 measured after TNF-αstimulation relative to untreated cells in the presence of AE (e.g.,n=3). Specifically, upon stimulation of the lung-on-chip microfluidicdevice 500 with TNF-α on the alveolar compartment 502A containing AE,the vascular ICAM-1 expression increases in a dose-dependent manner,reproducing endothelial stimulation as has been previously observed.

Referring to FIG. 29, and correspondingly with respect to the chart ofFIG. 28, the platelet-endothelial dynamics in the microfluidic device500 follows a similar trend as ICAM-1. Specifically, a chart showsplatelet-endothelial dynamics in an untreated vs TNF-α stimulatedlung-on-a-chip device 500 (n=3. *P<0.05).

Referring to FIG. 30, at the end of the assay, due to vascularactivation, significant amount of clots are observed. The clots areconstituted by platelets and fibrin localized within the vascularcompartment 502B of the lung-on-a-chip device 500. Specifically,fluorescent micrographs showing platelets (left), fibrin (middle), andmerged (right) on an endothelial surface, when stimulated by TNF-α(scale bar—100 μm). This confirms that the lung-on-a-chip device 500 issensitive to the pro-inflammatory effect of the TNFα. The epithelialstimulation, which can result in signalling from the epithelial side tothe endothelium, promotes activation of the endothelial cells and bloodcells, such as, platelets, finally causing intravascular thrombusformation.

C. Lipopolysaccharide (“LPS”) Induced Inflammation and Thrombosis

Referring generally to FIGS. 31-35, an evaluation is directed to morecomplex epithelial-endothelial-blood cell interactions and to betterdefine the link between local inflammation and thrombosis. Theevaluation includes stimulating a microfluidic device on the alveolarepithelial cells (AE) side with a lipopolysaccharide (LPS) endotoxin,and comparing P-E when the stimulation occurs in the presence or absenceof the alveolar epithelial cells (AE). The microfluidic device is any ofthe microfluidic devices described above (e.g., microfluidic device 300,lung-on-a-chip device 500, etc.).

Referring specifically to FIG. 31, a chart shows vascular ICAM-1 that ismeasured after LPS stimulation relative to untreated cells in thepresence or absence of the alveolar epithelial cells (AE) (n=3). Whenthe blood vessel alone is stimulated with LPS for 2 hours, it results inno significant increase in ICAM-1, and the endothelium is inflamed onlywhen the LPS stimulation occurs over the AE.

Referring specifically to FIG. 32, platelet-endothelial dynamicsmeasured in the microfluidic device, in the presence or absence of thealveolar epithelial cells (AE), are either left untreated or arestimulated with various doses of LPS (n=3, *P<0.001). Thus, the P-E isnear absent when the lung-on-a-chip device is left untreated or istreated with LPS in the absence of AE. Furthermore, the P-Esignificantly increases when the stimulation occurs over the AE.

Referring specifically to FIG. 33, representative fluorescencemicrographs show platelet aggregates and fibrin at the end of bloodperfusion through the microfluidic device. The microfluidic devicecontains and compares untreated and LPS stimulation, in the presence orabsence of AE (scale bar—100 μm). Thus, at the end of blood perfusion inthe LPS-stimulated microfluidic device containing AE, large plateletaggregates are formed along with significant fibrin in the lumen. Thisdemonstrates in vitro that in situ thrombosis in the vascular lumen iscaused by an LPS-directed epithelial injury.

Referring specifically to FIG. 34, a chart shows barrier permeabilitymeasured after LPS stimulation, relative to untreated cells in thepresence or absence of the alveolar epithelial cells (AE) (n=1 or 2).Thus, the trend that occurs due to the epithelium is further confirmedwhen the barrier permeability is measured.

Referring specifically to FIG. 35, representative confocal micrographsshow gap junctions under no treatment or LPS treatment, in the presenceof a blood vessel alone or with epithelium (AE). The visualized gapjunctions are not affected when the blood vessel is stimulated with LPS,but significantly increase in the presence of AE.

Referring generally to FIGS. 36 and 37, the technique described above inreference to FIGS. 31-35 has been further applied to a laser injuryinduced thrombus formation in vivo in a cremaster arteriole and acremaster vein of a mouse, along with a systemic delivery of LPS. When amouse is injured with laser, significant clotting is observed in boththe artery and vein, along with increase in P-E. The increase in P-E isfurther exacerbated when LPS is administered additionally. However,administration of LPS alone does not induce any platelet-endothelialinteractions.

Referring specifically to FIG. 36, fluorescent micrographs showevolution of blood clots (left to right) in a cremaster artery of themouse. The cremaster artery is left untreated, laser injured or aftersystemic injection of LPS (scale bar: 25 μm).

Referring specifically to FIG. 37, a chart shows platelet-endothelialdynamics computed on fluorescent time-series of platelets. The plateletsadhere to a cremaster artery or a vein of a mouse (n=3, *P<0.05).

Referring to FIG. 42, histological sections show sections of a mouselung with clots. The sections of the mouse lung are left untreated ortreated with LPS. Thus, in contrast to the results described inreference to FIGS. 36 and 27, the histological analysis of the mouselung shows that the LPS injection results in lung injury and pulmonarythrombosis. This shows that either an exogenous stimulation (e.g., alaser injury) or possibly an epithelium (e.g., in a lung), are likelyessential to platelet adhesion and thrombosis (as also recreated invitro) in the microfluidic device. Therefore, an LPS-stimulatedlung-on-a-chip device is potentially a robust model for testingpotential antithrombotic and anti-inflammatory drug candidates, in anorgan-level functional environment of pulmonaryepithelial-endothelial-blood cell signalling.

D. Analysis of Cytoprotective Effect of PAR-1 Inhibitor

Referring to FIG. 38, an illustration shows a microfluidic device 600that contains alveolar epithelial cells (AE) 602 treated with LPS 604and a vessel 606 treated with parmodulin (PM2) 608 to inhibit thrombosisdue to lung injury. Thus, the microfluidic device 600 is stimulated onthe AE side 602 with LPS and is perfused with blood 610 in the vessel606. The microfluidic device is similar to or identical with one or moreof the microfluidic devices described above. In response to thestimulation, no significant clotting occurs and P-E occurs PM2 is addedto the endothelial cell culture medium.

Referring to FIG. 39, a chart shows platelet-endothelial dynamics thatare measured in the microfluidic device 600 containing AE cells (n=3.*P<0.05,**P<0.01). Additionally, adding PM2 to blood 610 alone alsoresults in the reduction of P-E as it prevents PAR-1 activation ofplatelets. Furthermore, adding PM2 to both endothelium and plateletscompletely inhibits the P-E and thrombotic activity in thisLPS-stimulated alveolar microfluidic device 600.

As such, the approach described in reference to FIGS. 38 and 39 show acytoprotective and anti-thrombotic effect of PM2 in aphysiologically-relevant model of acute lung dysfunction. Thus, theapproach provides a beneficial drug candidate for intervention indiseases that cause pulmonary thrombosis. Notably, the approachdemonstrates the strength of the in vitro microfluidic device 600,allowing visualization and quantitative analysis of organ-levelinteractions in real-time, including the epithelium (e.g., stimulatedwith LPS), endothelium (e.g., protected by PM2), and whole blood cells(e.g., coagulation and platelet function). A similar experiment in vivois extremely difficult to perform, if not impossible.

IV. Algorithm—Discussion

Generally, a salient feature of pulmonary organ-on-chip microfluidictechnology is that it permits perfusion of human whole blood in itsnative state (e.g., recalcified after anticoagulation in sodium citrate)at any desirable shear rate. Thus, the microfluidic technology providesa significantly more physiologically-relevant in vitro platform to studyand analyze intravascular clotting of a lung organ.

Additionally, by harnessing the full potential of modern automatedfluorescence microscopy and mathematical algorithms that are designed toquantitate thrombus formation occurring in real-time inside amicrofluidic device, platelet-surface interactions are assessed overlarge spatiotemporal scales. The approaches described above and belowshow that the integrated interplay between platelets, thrombi, a vesselwall, blood-borne factors, and flow dynamics are analyzed in theintegrated system-level assay. Significantly, pulmonary clot formationis caused by endothelial activation that occurs in a microfluidic devicein vitro and correlates to a mouse laser injury model in vivo, both interms of morphology and regional heterogeneity. Thus, the describedmicrofluidic device and analytical methods potentially act as a valuabletool for analyzing organ-level tissue-tissue interactions underpathophysiological conditions that are relevant for thrombosis andplatelet research.

Furthermore, by incorporating primary alveolar epithelial cells that areco-cultured along with endothelial cells for up to 2 weeks, andmaintaining physiological junction integrity, a limitation of previouslung-on-a-chip devices (which contain tumor derived cell lines) has beenovercome. The present physiologically-relevant microfluidic deviceallows the finding that the epithelial cells can make a directcontribution to thrombosis, when stimulated by LPS. This finding is verydifficult to show in vivo in real-time, as individual tissue andcellular compartments inside an organ cannot be individually regulated,and blood flow inside lung vessels cannot be observed over a largesection.

Nevertheless, using the laser injury mouse model, it is demonstratedthat LPS does not cause thrombosis in a cremaster artery or vein, butcauses clotting in the lung. This is somewhat similar to in vitroobservations, where LPS stimulation of endothelium alone does not causethrombus formation, but, instead, LPS stimulation of epithelium resultsin vascular dysfunction and rapid clotting.

The described microfluidic devices and methods help unravel thecytoprotective and anti-thrombotic effect of a novel PAR-1 antagonist(PM2) in the setting of an acute lung injury and whole blood perfusion.The findings encourage the pharmaceutical industry to further testantithrombotic drugs using the described humanized platform, having thepotential to cause vasculopathy and bleeding as a major toxicity (whichis also very difficult to study in vivo). Overall, the describedpulmonary thrombosis-on-a-chip microfluidic device may be furtherbeneficial in finding potential applications in a variety of settingsrelevant for thrombosis research, e.g., toxicology, drug screening, anddiagnostics. One likely future benefit may permit a personalizedassessment of drug response to therapy, to help individualize drugdelivery, by using patient derived cells and blood.

V. Algorithm—Materials and Methods

A. Device Fabrication

According to one example, one or more of the microfluidic devicesdescribed above are fabricated with Prototherm 12120 usingstereolithography (such as provided by Protolabs, Maple Plain, Minn.).Top and bottom components of the microfluidic devices are cast from PDMSat a 10:1 w/w base to curing agent ratio. The components are degassedand, then, cured overnight for 4 hours at 60° C. The top componentcontains a fluidic channel (1×1 mm cross section) and ports for the topand bottom channels. PDMS membranes, which provide a semi-permeablebarrier between the epithelium and microvascular endothelium layers, arefabricated by casting against a DRIE-patterned silicon wafer (50×50 mm).The wafer has posts that are 50 μm high and have a 7 μm diameter. Theposts are spaced apart at a distance of 40 μm.

To produce through-holes in the membrane using the microfabricated postarray, after pouring 100 μL of PDMS onto the wafer, a polycarbonatebacking is compressed against the post array and is baked at 60° C. for4 hrs. The membrane is bonded to the top component using oxygen plasmatreatment (e.g., 40 Watts, 800 millibars, 40 seconds; Plasma Nano,Diener Electronic, Ebhausen, Germany), followed by bonding the assemblyof the top component and membrane to the bottom component containing anendothelial channel (1 mm wide×0.2 mm high). The microfluidic devicesare sterilized using oxygen plasma treatment (100 Watts, 15 standardcubic centimeters, 30-60 seconds; PlasmaEtcher PE-100, Plasma Etch,Reno, Nev.).

B. Cell Culture and Stimulation

After plasma treatment of the microfluidic devices, the two chambers arepre-treated with 10% (3-aminopropyl)-trimethoxysilane (APTMES, e.g.,from Sigma) in 100% anhydrous alcohol (e.g., from Sigma) for 10-20minutes. The chambers are flushed sequentially with 70% ethanol in waterand 100% ethanol, and then dried at 60-80° C. for two hours. Then, amixture of rat tail collagen I (100 μg ml⁻¹ in PBS, from BD Biosciences)and fibronectin (e.g., 30 μg/ml in PBSm, from BD Biosciences) isintroduced in both chambers of the microfluidic device. The microfluidicdevice is incubated at 37° C. for at least 2 hours before flushing withPBS or EGM-2 cell culture media.

Some of the microfluidic devices are used with collagen coating alone(without cells). In other devices, HUVECs (e.g. from Lonza) are culturedin Endothelial Growth Medium-2 (EGM-2, from Lonza) and used betweencertain passages. The cells (5-10×10⁶ cells/ml) are introduced into thecollagen-coated channels and incubated for 20 minutes at 37° C. topromote cell attachment before a second similar HUVEC suspension is,then, introduced. The microfluidic devices are incubated upside down foran additional 20 minutes to seed the cells on the ceiling and walls ofthe lower chamber.

The lower chamber is then flushed with EGM-2 and, then, a suspension ofprimary alveolar epithelial cells (e.g., from ScienCell Research Labs,Carlsbad, Calif.; 5-6×10⁶ cells ml⁻¹) is introduced into the top chamberof the device. After a few hours, the top and bottom chambers areflushed with their respective media, and the microfluidic devices areincubated at 37° C. under 5% CO₂ for 3 days (with media being replacedeach day once). On day 4, the bottom vascular chamber is set onperfusion of EGM-2 media (e.g., 30 μL hr⁻¹; 0.5% fetal bovine serum) toprovide shear to the HUVEC vessel chamber and a continuous supply offresh media.

On day 6, the epithelial cell media in the top chamber is aspirated tocreate the air-liquid interface of the alveoli. Hereon, the cell culturecontinues for another 6-8 days, after which, in some cases, the topchamber is supplemented with 0, 5, or 100 ng ml⁻¹ TNF-α in PBS (e.g.,from Sigma) overnight or LPS (e.g., 100 ng ml⁻¹ of E. Coli, from Sigma,)for 2 hours to cause cell activation. The LPS is sonicated in ultrasonicbath for about 20-30 minutes before introducing into the microfluidicdevice. After stimulation, the top chamber is clamped, the bottomchamber is rinsed with culture media, a reservoir cut from a 3 mlslip-tip syringe (e.g., from BD) is inserted on one end of the lowerchamber, and a 1/16-inch male luer connector (e.g., from Qosina Corp) isinserted on the other end of the lower chamber.

C. Blood Samples and Flow Conditions

Citrated human blood (e.g., from Research Blood Components, Cambridge,Mass.) is used within 5 hours of blood draw, to minimize pre-analyticaleffects on platelet function. Platelets are labeled with human CD41-PEantibody (e.g., 10 μl ml⁻¹, Invitrogen) that is directly added to theblood and is incubated at room temperature for about 10 minutes.

When analyzing the formation of fibrin, blood samples re added with 15μg ml⁻¹ of fluorescently labeled fibrinogen (e.g., Alexa 488 fromInvitrogen). The citrated blood is recalcified 2 minutes after thebeginning of each experiment by adding 100 μl mL⁻¹ of a solutioncontaining 100 mM calcium chloride and 75 mM magnesium chloride to theblood to permit calcium-dependent and magnesium-dependent plateletfunctions. Citrated human blood (e.g., 1.2 mL) is pipetted into a fluidreservoir fitted to one end of a microchannel on one side of themicrofluidic device.

A piece of medical grade tubing (e.g., 1.58 mm inner diameter of TygonS-50-HL from Saint Gobain Plastics) is fitted to the outlet port of thedevice via a barbed luer lock connector (e.g., Harvard Apparatus). Theother end of the tube is connected to a 3 ml syringe (e.g., from BectonDickinson) through which blood is withdrawn from the device by pullingthe blood using a syringe pump (e.g., PHD Ultra CP, Harvard Apparatus),thereby driving blood flow through the microfluidic device. The flowrate is adjusted to result in a wall shear rate of 250 sec⁻¹ (e.g.,approximately 10 dynes/cm² stress). For studying platelet-endothelialdynamics at a higher wall shear rate of 750 sec⁻¹ (e.g., approximately30 dynes/cm² stress), a microfluidic device has an endothelial chambersmaller in size (e.g., 0.4 mm wide×0.1 mm high) to facilitateexperiments in which less than 1 ml of blood is used.

D. Image Acquisition and Analysis

Platelet dynamics are visualized using time-lapse fluorescence imaging(e.g., LD Plan Neofluar 10×, NA 0.4; Zeiss Axio Observer; Hamamatsu ORCAC11440 CMOS digital camera) using an exposure time of 200 ms. Images aretiled to create a composite panoramic view (e.g., 18,600 pixels long and2,050 pixels wide; 1 pixel=0.325 μm). Images are archived as OME-TIFFformat files, and an image analysis is performed using, for example,Zeiss Zen 2012 imaging software and MATLAB 2014 routines.

E. Platelet-Endothelial Dynamics Algorithm

Referring generally to FIGS. 43-50, illustrations show the visualizationand analysis of platelet-endothelial temporal dynamics. Specifically,the illustrations include space-time kymographs that are plotted toillustrate acquired image time-series and graphed fluorescence at arepresentative pixel over time, after removing the (linear) trend due tothe increasing platelet adhesion alone.

Referring specifically to FIGS. 43-46, representative kymographs of asmall section of a microchannel show an attachment and detachmentpattern of platelets on a collagen surface (FIG. 43) or vessel that isuntreated (FIG. 44) or TNF-α treated (FIG. 45). Referring specificallyto FIG. 46, the coefficient of variance (CV) of a fluorescence signal isobserved over time at a representative single pixel location of an imagetime-series of platelet accumulation, as plotted in the kymographs shownin FIGS. 43-45.

No significant fluctuations are shown on both the collagen-coatedmicrofluidic surface (FIG. 43) and the surface lined with a healthy liveendothelium (FIG. 44). Thus, on collagen, while there is plateletaccumulation over time, there is hardly any detachment or dynamicalsurface interactions present. A healthy endothelium is devoid of anyinteraction at all. However, TNF-α stimulated endothelium results insignificant fluctuations (FIG. 45), which is representative ofoccasional attachment and detachment of platelets (dynamics).

To define a parameter that quantitates these differences at a pixellevel, a dimensionless statistical measure of dispersion is selected asa coefficient of variance (CV). The CV is defined as a ratio of standarddeviation and mean. Although the CV at a representative pixel is nearzero for collagen and untreated endothelium, as FIG. 46 illustrates theCV is much higher for a treated endothelium.

Referring specifically to FIGS. 47-50, a visualization and analysis ofplatelet-endothelial spatial dynamics is illustrated. Top images arerepresentative CV-colormaps of a large section of the vessel where eachpixel in the map represents the temporal platelet dynamics (CV) on acollagen surface or vessel (untreated vs TNF-α treated, with a scalebar: 100 μm). Bottom graphs show CV across the length of the channel atrepresentative width location for collagen (FIG. 47), untreated vessel(FIG. 48), and vessel treated with TNF-α (FIG. 49). The dotted lines aredrawn at the 95^(th) percentile and 5^(th) percentile value of the CVrespectively. The graph of FIG. 50 illustrates the interpercentile range(95^(th)-5^(th) percentile value) of the CV plotted in the graphsillustrated in FIGS. 47-49, as a measure of depicting spatialheterogeneity in platelet accumulation.

FIGS. 47-50 are directed to a much larger area of the lumen, creating a2-D image/map of the CV of all the pixels of the acquired imagetime-series. The resulting spatial image is reprocessed using a colormap and is analysed using an intensity palette look-up table to contrasthighly active versus dormant areas. This image intensity transformationenables spatial visualisation of dynamic behaviour of individualplatelets, in addition to conveying an overall pattern of thrombidynamics.

For example, FIG. 47 shows a uniform platelet adhesion pattern with anarrow range of variance on a cell free collagen surface. However, inFIG. 48, when blood is flowed over a healthy endothelium, platelets showvery limited reactivity with the apical surface and, therefore, thecolor spectrum is almost entirely black. Nevertheless, in FIG. 49, thethrombi patterns on endothelium treated TNF-α are heterogeneous andfluctuate significantly.

Furthermore, FIG. 50 is representative of a visual analysis of the localheterogeneity within the thrombus. A plot of the CV, over the entirelength of the microchannel, at a representative location along the widthof the channel, provides the spatial heterogeneity or fluctuations ofthe dispersion parameter (CV) for a collagen coated device or anendothelium. The interpercentile range (e.g., the difference between the95^(th) percentile and 5^(th) percentile) shows that the spatialheterogeneity also varies between the surfaces. On collagen and ahealthy endothelium, there is very little variation between thesurfaces. In contrast, the spatial heterogeneity is very high on astimulated endothelium. Equation 1, described above, shows thederivation of the quantitative readout as a combination of CV andinterpercentile range.

F. Immunostaining and Histology

Fluorescence microscopy is optionally performed on an endothelium thatis fixed with 4% formaldehyde (from Sigma) and stained with antibodiesagainst ICAM-1 (from Santa Cruz) and VE-Cadherin (from Santa Cruz). Theendothelium is further counterstained with phalloidin and DAPI (fromInvitrogen).

G. Parmodulin (“PM2”) Drug Delivery

PM2 is optionally added to the endothelial cell or epithelial cellculture medium at a final concentration of 30 μM. The cells are exposedfor about 4 hours. Then, the cells are stimulated with LPS (100 ng ml⁻¹)for about 2 hours. LPS also contains PM2 (30 μM). Blood is, then,perfused in the microfluidic device. In whole blood containing PM2, theblood is added to a final concentration of 30 μM, and is incubated forabout 30 minutes before perfusion.

H. Mouse Laser Injury Model

In the above described techniques, C57BL/6J mice (about 8-12 weeks old)are used. For example, the mice are purchased from the JacksonLaboratory (Bar Harbor, Me.). Animal care and experimental proceduresare performed in accordance with and under approval of the Beth IsraelDeaconess Medical Center (“BIDMC”) Institutional Animal Care and UseCommittee.

LPS is isolated from Escherichia coli serotype 0111:B4 (e.g., fromSigma-Aldrich, St Louis, Mo.). The anti-platelet antibody CD42bconjugated to Dylight649 is purchased, for example, from EmfretAnalytics (Eibelstadt, Germany).

A laser-induced injury model of thrombosis is optionally used to monitorthrombosis formation in cremaster arterioles and venules in response tointraperitoneal injection of LPS (e.g., 10 mg kg′) or vehicle (e.g.,physiological saline solution). Intravital microscopy of the cremastermicrocirculation is performed, with injury to a cremaster arteriolar(e.g., 30-50 μm diameter) vessel wall being induced with a MicropointLaser System (e.g., from Photonics Instruments, Chicago, Ill.). TheMicropoint Laser System is focused through a microscope objective,parfocal with the focal plane and tuned to 440 nm. Data is captureddigitally in a single fluorescence channel at 647/670 nm. Dataacquisition is initiated both prior to and following a single laserpulse for each injury. Images are captured using a CCD camera (e.g.,from Hamamatsu) at frame rates of 1/0.2 s⁻¹ and 1/0.5 s⁻¹, for a totalof 240 seconds. The microscope system is controlled and images areanalyzed using Slidebook (e.g., from Intelligent Imaging Innovations,Denver, Colo.). Anti-platelet antibodies re infused into the mice priorto vessel wall injury.

Histology Lungs are harvested in 4% paraformaldehyde. Followingovernight incubation, lungs re transferred to 70% ethanol.Paraffin-embedded lungs are sectioned and stained with Hematoxylin andeosin (MTS) or Masson's Trichome stain (MTS). This work is done by thehistology and microscopy core at BIDMC.

I. Statistical Analysis

Unless otherwise specifically mentioned above or in the drawings, alldata is presented as mean±standard error of the mean (SEM). Two-tailed Pvalues are obtained from the statistical t-test or analysis of variance(ANOVA) to compare the means. Data analysis is optionally performedusing Graphpad Prism V6.

VI. Other Embodiments

A. Organ-On-Chip (OOC) Device

Referring to FIGS. 51 and 52, a microfluidic system 700 is configured tofunction in accordance with one or more of the above-describedtechniques. According to the illustrated example, the microfluidicsystem 700 includes an organ-on-chip (“OOC”) device 710. The OOC device710 that includes a body 712 that is typically made of a polymericmaterial. The body 712 includes a first fluid inlet 714 a and a firstfluid outlet 714 b. The body 712 further includes a second fluid inlet716 a and a second fluid outlet 716 b. The first fluid inlet 714 a andthe first fluid outlet 714 b allow fluid flow through a firstmicrochannel 724. The second fluid inlet 716 a and the second fluidoutlet 716 b allow fluid flow through a second microchannel 726.

The first microchannel 724 is separated from the second microchannel 726by a barrier 730. The barrier 730 may be any suitable semi-permeablebarrier that permits migration of cells, particulates, media, proteins,and/or chemicals between the first microchannel 724 and the secondmicrochannel 726. For example, the barrier 730 includes gels, layers ofdifferent tissue, arrays of micro-pillars, membranes, combinationsthereof, and the like. Depending on the application, the barrier 730 mayhave openings or pores to permit the migration of cells, particulates,media, proteins, and/or chemicals between the first microchannel 724 andthe second microchannel 726. In some preferred embodiments, the barrier730 is a porous membrane that includes a cell layer 734 disposed on atleast a first surface of the membrane.

Optionally or alternatively, the barrier 730 includes more than a singlecell layer 734 disposed thereon. For example, the barrier 730 includesthe cell layer 734 disposed within the first microchannel 724, thesecond microchannel 726, or each of the first and second microchannels724, 726. Additionally or alternatively, the barrier 730 includes afirst cell layer 734 disposed within the first microchannel 724 and asecond cell layer within the second microchannel 726. Additionally oralternatively, the barrier 730 includes a first cell layer 734 and asecond cell layer disposed within the first microchannel 724, the secondmicrochannel 726, or each of the first and second microchannels 724,726. In one embodiment of the OOC device 710, the first and secondmicrochannels 724, 726 generally have a length of less than about 2 cm,a height of less than 200 μm, and a width of less than 400 μm. Moredetails on the OOC device 710 can be found in, for example, U.S. Pat.No. 8,647,861, which is owned by the assignee of the present applicationand is incorporated by reference in its entirety.

Referring to FIG. 53, the barrier 730 includes pores 731, which can havevarious dimensions based on the barrier 730 that is chosen. In theillustrated example, a cell layer 734 is disposed within the firstmicrochannel 724 and on the first upper surface of the barrier 730.Fluid enters the first microchannel 724 and flows from the inlet towardthe outlet of the first microchannel 724. As the fluid flows from theinlet toward the outlet of the first microchannel 724, contact betweenthe fluid and the surface of the cells 734 exerts a shear stress on thecells 734. This shear stress deform the individual cells 734, or affectother changes in the physical or biological properties of the cells 734.

B. Exemplary Method for Determining Thrombosis Function

In accordance with the exemplary microfluidic system 700 (FIGS. 51-53),and the above-discussed techniques, an exemplary method is directed todetermining a thrombosis function. The method includes flowing a fluidsample 800 over a top surface of the membrane 730, which includes aendothelial cell monolayer 734. The method further includes stimulatingthe fixed endothelial cell monolayer 734 to induce formation of a clot802, the clot being formed via an interaction between the fixedendothelial cell monolayer 734 and the fluid sample 800. In response tothe clot formation, a thrombosis function is determined that isassociated with the fluid sample 800 and the fixed endothelial cellmonolayer 734.

In accordance with an alternative embodiment, in reference to the aboveexemplary method, the fluid sample 800 includes platelets 804 thatinteract with the fixed endothelial cell monolayer 734. In this example,the thrombosis function is a function of platelets in the fluid sample800.

In accordance with an alternative embodiment, in reference to the aboveexemplary method, the fixed endothelial cell monolayer is derived fromone or more of (a) a fixing endothelial cell extract, (b) endothelialcell-associated proteins that are adhered to the surface, (c) a subjectfrom which the fluid sample 800 is derived, (d) a subject that isdifferent than a subject from which the fluid sample 800 is derived, (e)fixing an endothelial cell monolayer 734 that has been grown on thesurface for a period of time, (f) healthy cells, and (g) diseased cells.

In accordance with an alternative embodiment, in reference to the aboveexemplary method, the fluid sample 800 includes one or more of a bloodsample, a serum sample, a plasma sample, a lipid solution, and anutrient medium.

In accordance with an alternative embodiment, in reference to the aboveexemplary method, the endothelial cell monolayer 734 is physically fixedby one or more of (a) exposing to air, (b) washing with alcohol,acetone, or a solvent that removes water, or lipids, (c) a chemicalfixative, (d) a decellularization solvent that stabilizes surfacemembrane protein configuration and cytoskeleton of a cell, (e) drying,and (f) dehydration.

In accordance with an alternative embodiment, in reference to the aboveexemplary method, the chemical fixative is selected from a groupconsisting of formaldehyde, paraformaldehyde, formalin, glutaraldehyde,mercuric chloride-based fixatives, precipitating fixatives, and dimethylsuberimidate.

In accordance with an alternative embodiment, in reference to the aboveexemplary method, the method further includes measuring at least one oftemporal and spatial interaction dynamics of cells in the fluid sample.

In accordance with an alternative embodiment, in reference to the aboveexemplary method, the cells are platelets and the spatial interactiondynamics of the cells includes at least one of (a) binding dynamics ofthe platelets to the fixed endothelial cell monolayer and (b) bindingdynamics of the platelets to each other.

In accordance with an alternative embodiment, in reference to the aboveexemplary method, the method further includes storing the top surface ofthe membrane 730 at (a) room temperature for a predetermined period oftime prior to said flowing the fluid sample 800 or (b) a temperature ofabout 4° C. or lower for a predetermined period of time prior to theflowing of the fluid sample 800.

In accordance with an alternative embodiment, in reference to the aboveexemplary method, the flowing of the fluid sample 800 is at (a) aphysiological shear rate, (b) a pathological shear rate, or (c) at ashear rate of about 50 sec⁻¹ to about 10,000 sec⁻¹.

C. Exemplary Microfluidic System for Determining Thrombosis Function

In accordance with another alternative embodiment, the microfluidicsystem 700 is directed to determining a thrombosis function and includesa compartment in the form of the first microchannel 724. The membrane730 has the fixed endothelial cell monolayer 734 on the top surface ofthe membrane 730. As such, the compartment 724 is configured to receivethe fluid sample 800 flowing over the top surface of the membrane 730such that cells in the fluid sample 800 interact with the fixedendothelial cell monolayer 734.

The microfluidic system 700 further includes a detection module 810 thatis configured to detect interaction between the cells of the fluidsample 800 and the fixed endothelial cell monolayer 734. Additionally,the detection module 810 is configured to detect a function of the cellsin the fluid sample 800.

In accordance with an alternative embodiment, in reference to the aboveexemplary microfluidic system 700, the compartment 724 includes amembrane 730 having a top surface and a bottom surface. The firstmicrochannel 724 is a top microchannel that is separated from the secondmicrochannel 726, which is a bottom microchannel, by the membrane 730.

In accordance with an alternative embodiment, in reference to the aboveexemplary microfluidic system 700, the fluid sample 800 interact withany internal surfaces of the compartment 724 or the top surface of themembrane 730. Alternatively or additionally, the fluid sample 800 flowsthrough the bottom microchannel 726 and the fluid sample 800 interactwith any internal surface of the compartment 726 or the bottom surfaceof the membrane 730.

In accordance with an alternative embodiment, in reference to the aboveexemplary microfluidic system 700, the top surface of the membrane 730includes the fixed endothelial cell monolayer 734, and the bottomsurface of the membrane 730 including adhered tissue-specific cells 814.

In accordance with an alternative embodiment, in reference to the aboveexemplary microfluidic system 700, the detection module 812 includes animaging system 816 configured to provide images of interaction betweenthe cells of the fluid sample 800 and the fixed endothelial cellmonolayer 734.

In accordance with an alternative embodiment, in reference to the aboveexemplary microfluidic system 700, the imaging system 816 includes atime-lapse microscopy apparatus 818.

In accordance with an alternative embodiment, in reference to the aboveexemplary microfluidic system 700, the detection module 812 includes oneor more of a wide-field holography apparatus 820, an impedancespectroscopy apparatus 822, a flow sensor apparatus 824, and a pressuresensor apparatus 826.

In accordance with an alternative embodiment, in reference to the aboveexemplary microfluidic system 700, the cells in the fluid sample 800include platelets, the detection module 812 being configured todetermine a function of the platelets in the fluid sample 800.

D. Exemplary System and Method for Quantifying Thrombosis

In accordance with another alternative embodiment, the microfluidicsystem 700 is a system for quantifying thrombosis in vitro based onphysiological conditions. By way of example, the membrane 730 is in theform of a solid substrate having a top surface with the fixedendothelial cell monolayer 734. The microfluidic system 700 includes thedetection module 812, which is configured to receive the solid substrate730 and to detect spatial and temporal interaction between cells in thefluid sample 800 and the surface of the solid substrate 730 when thefluid sample 800 is flowed over the surface along a flow axis F.

The system 700 includes one or more controllers 830 that are configuredto store time-lapse data of detectable signals collected from thedetection module 812, the detectable signals representing spatial andtemporal interaction between the cells of the fluid sample 800 and thesurface of the fixed endothelial cell monolayer 734. The controllers 830generate a kymograph from at least a portion of the stored time-lapsedata, wherein a time axis of the kymograph indicates at least a portionof the time-lapse duration, a space axis of the kymograph indicating thedetectable signals along the flow axis.

Based on the generated kymograph, the controllers 830 determine a rateof fluctuation in a coefficient of variation (CV) of the detectablesignals to generate a temporal cell dynamics index. The controllers 830further determine either (i) the presence of reactive cells in the fluidsample 800 when the temporal cell dynamics index is higher than atemporal control value, or (ii) the absence of reactive cells in thefluid sample 800 when the temporal cell dynamics index is no more thanthe temporal control value

The system 700 further includes a display module 840 for displayingcontent that is based in part on output determined by the one or morecontrollers 830, wherein the content includes a signal indicative ofeither presence or absence of at least one of reactive cells or cellaggregation in the fluid sample 800.

In accordance with an alternative embodiment, in reference to the aboveexemplary system 700, the fluid sample 800 is blood and the cells areplatelets.

In accordance with an alternative embodiment, in reference to the aboveexemplary system 700, the detectable signals are averaged across a widthof the surface prior to generating the kymograph.

In accordance with an alternative embodiment, in reference to the aboveexemplary system 700, the width of the surface is transverse to the flowaxis.

In accordance with an alternative embodiment, in reference to the aboveexemplary system 700, the controllers 830 are further configured togenerate, from at least a portion of the stored time-lapse data, aspatial map of temporal variances of the detectable signals, each pixelof the spatial map corresponding to a time-averaged CV of the detectablesignals.

In accordance with an alternative embodiment, in reference to the aboveexemplary system 700, the controllers 830 are further configured todetermine, based on the generated spatial map, an inter-quartile range(IQR) of the map to generate a spatial cell dynamics index.

In accordance with an alternative embodiment, in reference to the aboveexemplary system 700, the controllers 830 are further configured todetermine the presence of cell aggregation in the fluid sample 800 whenthe spatial platelet dynamics index is higher than a spatial controlvalue, and the absence of cell aggregation in the fluid sample 800 whenthe spatial platelet dynamics index is no more than the spatial controlvalue.

In accordance with an alternative embodiment, in reference to the aboveexemplary system 700, the time-lapse data is presented in the form ofimages.

In accordance with an alternative embodiment, in reference to the aboveexemplary system 700, the controllers 830 are further configured todetermine cell reactivity based on a linear or non-linear function thatincludes spatial and temporal dynamic parameters.

In accordance with an alternative embodiment, a method is directed toquantifying thrombosis in vitro based on physiological conditions inaccordance with the above exemplary system 700.

VII. Exemplary Embodiments A. Displayed Content of Display Module

In some embodiments, referring to FIG. 62, depending on the nature ofthe fluid samples and/or applications of the systems as desired byusers, the display module 908, 1108 can further display additionalcontent. In some embodiments where the fluid sample is collected orderived from a subject for diagnostic assessment, the content displayedon the display module 908, 1108 can further comprise a signal indicativeof a diagnosis of a condition (e.g., disease or disorder) or a state ofthe condition (e.g., disease or disorder) in the subject. For example,in some embodiments where the subject is diagnosed for plateletdysfunction, the content can further comprise a signal indicative of adisease or disorder induced by platelet dysfunction. Examples of thedisease or disorder induced by platelet dysfunction can include, but arenot limited to thrombosis, an inflammatory vascular disease (e.g.,sepsis, or rheumatoid arthritis), a cardiovascular disorder (e.g., acutecoronary syndromes, stroke, or diabetes mellitus), vasculopathies (e.g.,malaria, disseminated intravascular coagulation), or a combination oftwo or more thereof.

In some embodiments wherein the fluid sample is collected or derivedfrom a subject for selection and/or evaluation of a treatment regimenfor a subject, the content can further comprise a signal indicative of atreatment regimen personalized to the subject, based on the computedtemporal cell (e.g., platelet) dynamic index and/or spatial cell (e.g.,platelet) dynamic index, as compared to a corresponding control value(e.g., based on healthy subjects, or from the same subject before theonset of the treatment regimen, or at an earlier time point of thetreatment regimen).

The methods and/or systems described herein can provide tools todiagnose a disease or disorder induced by cell dysfunction and/orabnormal cell-cell interaction in a subject. Accordingly, another aspectdescribed herein relates to a method of determining if a subject is atrisk, or has, a disease or disorder induced by cell dysfunction orabnormal cell-cell interaction. The method comprises: (a) flowing afluid sample of the subject over a surface comprising a fixed cellmonolayer thereon; (b) detecting interaction of cells in the fluidsample between each other and/or with the fixed cell monolayer; and (d)identifying the subject to be at risk, or have the disease or disorderinduced by cell dysfunction when the cell-cell interaction is higherthan a control; or identifying the subject to be less likely to have adisease or disorder induced by cell dysfunction when the cell-cellinteraction is no more than the control.

In some embodiments, the fixed cell monolayer used in the methodsdescribed herein can be subject-specific.

In some embodiments, the method of determining if a subject is at risk,or has a disease or disorder induced by cell dysfunction and/or abnormalcell-cell interaction can be used for diagnosis and/or prognosis of adisease or disorder induced by blood cell dysfunction (e.g., plateletdysfunction), and/or guiding and/or monitoring of an anti-plateletand/or anti-inflammation therapy. Accordingly, in some embodiments, thefixed endothelial cell monolayer can comprise a fixed endothelial cellmonolayer. The fixed endothelial cell monolayer can be subject-specific.In some embodiments, the fluid sample can comprise blood cells such asplatelets. Thus, a method of determining if a subject is at risk, or hasa disease or disorder induced by blood cell dysfunction (e.g., plateletdysfunction) is also described herein. Non-limiting examples of thedisease or disorder induced by blood cell dysfunction (e.g., plateletdysfunction) include, but are not limited to thrombosis, an inflammatoryvascular disease (e.g., sepsis, or rheumatoid arthritis), acardiovascular disorder (e.g., acute coronary syndromes, stroke, ordiabetes mellitus), vasculopathies (e.g., malaria, disseminatedintravascular coagulation), or a combination of two or more thereof. Inthese embodiments, the method can further comprising administering tothe subject identified to at risk or has the disease or disorder inducedby blood cell dysfunction (e.g., platelet dysfunction) an appropriatetreatment (e.g., anti-platelet therapy, or an anti-inflammationtherapy).

B. Compositions for Determining Cell-Cell Interaction

Compositions for determining cell-cell interaction are also describedherein. In one aspect, the composition comprises (a) a solid substratehaving a surface comprising a monolayer of cells of a first typethereon; and (b) a fluid sample in contact with the surface, wherein thefluid sample comprises cells of a second type.

In some embodiments, the monolayer of cells of the first type cancomprise a fixed endothelial cell monolayer. In some embodiments, thecells of the second type in the fluid sample can comprise blood cellssuch as platelets.

In some embodiments, the fluid sample can comprise a blood sample.

The cell monolayer can comprise fixed cells (e.g., fixed endothelialcells), fixed cell extract(s) (e.g., fixed endothelial cell extract(s)),and/or fixed cell-associated proteins (e.g., fixed endothelialcell-associated proteins) that are adhered to the surface.

In some embodiments, the cell monolayer (e.g., endothelial cellmonolayer) can be derived from fixing a cell layer (e.g., an endothelialcell monolayer) that has been grown on the surface for a period of time,e.g., until the cell layer reaches confluence.

The surface with which the fluid sample is in contact can be a surfaceof any fluid-flowing conduit disposed in a solid substrate. The solidsubstrate can be any solid substrate that is compatible to the fluidsample and the cell monolayer. Non-limiting examples of the solidsubstrate include a cell culture device, a microscopic slide, a cellculture dish, a microfluidic device, a microwell, and any combinationsthereof.

In one embodiment, the surface can be a wall surface of a microchannel.In one embodiment, the surface can be a surface of a membrane. In someembodiments where the surface is a surface of a membrane, the membranecan be configured to separate a first chamber (e.g., a firstmicrochannel) and a second chamber (e.g., a second microchannel) in amicrofluidic device.

In some embodiments, the microfluidic device can be configured tocomprise an organ-on-a-chip device as described herein. An exemplaryorgan-on-chip can comprise a first chamber (e.g., a first microchannel),a second chamber (e.g., a second microchannel), and a membraneseparating the first chamber and the second chamber. In theseembodiments, a first surface of the membrane facing the first chambercan comprise the cell monolayer (e.g., endothelial cell monolayer)thereon, and a second surface of the membrane facing the second chambercan comprise tissue-specific cells adhered thereon.

C. Additional Example of Applications of the Methods, Systems, andCompositions Described Herein

The methods, systems, and compositions of various aspects describedherein can be used to determine cell-cell interaction, e.g., but notlimited to spatial and/or temporal dynamics of cells of a first typeinteracting with each other or with cells of a second type. In someembodiments, the methods, systems, and compositions of various aspectsdescribed herein can be used to determine blood cell dynamics (e.g.,platelet dynamics).

For example, in some embodiments, the methods, systems, and/orcompositions described herein can be configured to permit a bloodcell-comprising fluid sample (e.g., platelet-comprising fluid sample)flowing over a more reliable and physiologically relevantendothelialized surface inflamed by a cytokine, thus mimicking the invivo endothelium-blood cell (e.g., platelet) crosstalk environment,e.g., in a normal or diseased state. The blood cell (e.g., platelet)dynamics (e.g., adhesion, translocation and/or detachment) can berecorded and quantified, which is not possible with the existing goldstandard tests. As the blood cell (e.g., platelet) function/interactioncan be reproduced even when the live endothelial cells are fixed, thecompositions with a fixed endothelial cell monolayer described hereincan be stored under standard laboratory conditions for a period of time(e.g., days or weeks) and still remain functional. Thus, thecompositions described herein can be operated near patients' bedside,e.g., in clinics or hospitals, to determine blood cell (e.g., platelet)dysfunction, e.g., for diagnosis of a disease or disorder induced byblood cell (e.g., platelet) dysfunction.

In some embodiments, the compositions described herein can furthercomprise tissue-specific cells. For example, in some embodiments, amicrofluidic device can comprise a first chamber (e.g., a firstmicrochannel), a second chamber (e.g., a second microchannel), and amembrane separating the first chamber and the second chamber, wherein afirst surface of the membrane facing the first chamber can comprise aendothelial cell monolayer thereon, and a second surface of the membranefacing the second chamber can comprise tissue-specific cells adheredthereon. A fluid comprising blood cells (e.g., blood or bloodsubstitute) can be introduced into the first chamber such that bloodcells can interact with the endothelial cell monolayer. In someembodiments, the endothelial monolayer can be an inflamed or diseasedendothelial cell monolayer. By incorporating luminal blood cell fluidtransport (e.g., a fluid comprising blood cells such as platelets) overa fixed endothelial cell monolayer and live culture of tissue specificcells, a physiologically relevant in vitro model of blood cell-inducedinflammation can be created to probe its pathophysiology and/or topermit drug screening.

Accordingly, in one aspect, a method for modeling a blood cell-induceddisease or disorder in vitro is also described herein. Examples of ablood cell-induced disease or disorder can include, but are not limitedto, thrombosis, an inflammatory vascular disease (e.g., sepsis, orrheumatoid arthritis), a cardiovascular disorder (e.g., acute coronarysyndromes, stroke, or diabetes mellitus), vasculopathies (e.g., malaria,disseminated intravascular coagulation), or a combination of two or morethereof. The method comprises flowing a fluid sample comprising diseasedblood cells (e.g., red blood cells, white blood cells, and/or platelets)over a surface comprising an endothelial cell monolayer (endothelium) ina cell or tissue culture device; and detecting interaction between theblood cells in the fluid sample and the endothelium, e.g., using theanalytical methods and/or systems described herein to determine dynamicsof blood cells binding to each other and/or to the endothelium. In someembodiments, the endothelium can be a normal endothelium. In someembodiments, the endothelium can be an inflamed endothelium.

The endothelium can comprise living endothelial cells or can be fixed asdescribed herein. In some embodiments where a fixed endothelium is used,the disease state is induced in the endothelium prior to fixation. Insome embodiments, a fixed endothelium can be used to model a diseasewhen the disease state is a result of components in the blood that donot act on the endothelium.

In some embodiments, the diseased blood cells and/or endothelial cellscan be collected from a subject diagnosed with a blood cell-induceddisease or disorder.

In some embodiments, diseased blood cells and/or endothelial cells canbe differentiated from induced pluripotent stem cells derived frompatients carrying a blood cell-induced disease or disorder. The diseasedblood cells and/or endothelial cells can then be manipulated, e.g.,using genome engineering technologies such as CRISPRs (clusteredregularly interspaced short palindromic repeats), to introduce orcorrect mutations present in the cells.

In some embodiments where normal, healthy blood cells and/or endothelialcells are used, the blood cells and/or endothelial cells can becontacted with an agent (e.g., an inflammation-inducing agent asdescribed herein) that induces the blood cells and/or endothelial cellsto acquire at least one phenotypic characteristic associated with ablood cell-induced disease or disorder.

In another aspect, a method for assessing blood substitute is alsodescribed herein. The method comprising flowing a blood substitute overa surface comprising an endothelial cell monolayer (endothelium) in acell or tissue culture device, and detecting interaction between theblood cells in the fluid sample and the endothelium; and determiningtemporal and/or spatial dynamics of blood substitute cells binding toeach other and/or to the endothelium using the analytical methods and/orsystems described herein.

As used herein, the term “blood substitute” is a substitute for blood,which has the ability to transport and supply oxygen to cells.

In a further aspect, a method for screening for agent(s) to reduce atleast one phenotypic characteristics of blood cell dysfunction (e.g.,platelet dysfunction) is also described herein. The method comprises (a)flowing a fluid sample comprising diseased blood cells (e.g., increasedcell adhesion to an endothelium and/or aggregation) over a surfacecomprising an endothelial cell monolayer (endothelium) in a cell ortissue culture device; (b) contacting the diseased blood cells and/orendothelium with a library of candidate agents; and (c) detectingresponse of the diseased blood cells and/or endothelium to the candidateagents to identify agent(s) based on detection of the presence of areduction (e.g., by at least about 30% or more) in the phenotypiccharacteristic of blood cell dysfunction (e.g., platelet dysfunction).

In some embodiments, the endothelium can be a normal endothelium. Insome embodiments, the endothelium can be an inflamed endothelium.

The candidate agents can be selected from the group consisting ofproteins, peptides, nucleic acids (e.g., but not limited to, siRNA,anti-miRs, antisense oligonucleotides, and ribozymes), small molecules,and a combination of two or more thereof.

Effects of the candidate agents on the diseased blood cells and/orendothelium can be determined by measuring response of the cells andcomparing the measured response with cells that are not contacted withthe candidate agents. Various methods to measure cell response are knownin the art, including, but not limited to, cell labeling,immunostaining, optical or microscopic imaging (e.g., immunofluorescencemicroscopy and/or scanning electron microscopy), spectroscopy, geneexpression analysis, cytokine/chemokine secretion analysis, metaboliteanalysis, polymerase chain reaction (PCR), immunoassays, ELISA, genearrays, spectroscopy, immunostaining, electrochemical detection,polynucleotide detection, fluorescence anisotropy, fluorescenceresonance energy transfer, electron transfer, enzyme assay, magnetism,electrical conductivity (e.g., trans-epithelial electrical resistance(TEER)), isoelectric focusing, chromatography, immunoprecipitation,immunoseparation, aptamer binding, filtration, electrophoresis, use of aCCD camera, mass spectroscopy, or any combination thereof. Detection,such as cell detection, can be carried out using light microscopy withphase contrast imaging and/or fluorescence microscopy based on thecharacteristic size, shape and refractile characteristics of specificcell types.

In some embodiments, a first surface of the membrane facing the firstchannel comprises an endothelium adhered thereon. In some embodiments, asecond surface of the membrane facing the second channel can comprisetissue-specific cells adhered thereon. As used herein, the term“tissue-specific cells” refers to parenchymal cells (e.g., epithelialcells) derived from a tissue or an organ, including, e.g., but are notlimited to, lung, brain, nerve network, blood-brain-barrier, kidney,liver, heart, spleen, pancreas, ovary, testis, prostate, skin, eye, ear,skeletal muscle, colon, intestine, and esophagus. By way of exampleonly, platelets have been contemplated to play a central role in avariety of inflammatory vascular diseases, such as sepsis, rheumatoidarthritis etc. and other vasculopathies that may involve endothelialbarrier dysfunction, such as malaria, where lung or brain are involved.Accordingly, in some embodiments, the second surface of the membranefacing the second channel can comprise lung cells or brain cells (e.g.,astrocytes) to create an in vitro model of malaria that incorporatesblood transport and endothelial barrier function.

D. Exemplary Fluid Sample

In accordance with various aspects described herein, a fluid sample(processed or unprocessed) comprising target cells to be analyzed can besubjected to the methods and systems described herein. In someembodiments, the fluid sample can comprise a biological fluid obtainedfrom a subject. Exemplary biological fluids obtained from a subject,e.g., a mammalian subject such as a human subject or a domestic pet suchas a cat or dog, can include, but are not limited to, blood (includingwhole blood, plasma, cord blood and serum), lactation products (e.g.,milk), amniotic fluids, sputum, saliva, urine, semen, cerebrospinalfluid, bronchial aspirate, perspiration, mucus, liquefied feces,synovial fluid, lymphatic fluid, tears, tracheal aspirate, and fractionsthereof.

In some embodiments, the biological fluid sample can comprise a bloodsample, a serum sample, a plasma sample, a lipid solution, a nutrientmedium, or a combination of two or more thereof.

The biological fluid sample can be freshly collected from a subject or apreviously collected sample. In some embodiments, the biological fluidsample used in the methods and/or systems described herein can becollected from a subject no more than 24 hours, no more than 12 hours,no more than 6 hours, no more than 3 hours, no more than 2 hours, nomore than 1 hour, no more than 30 mins or shorter.

In some embodiments, the biological fluid sample or any fluid sampledescribed herein can be treated with a chemical and/or biologicalreagent described herein prior to use with the methods and/or systemsdescribed herein. In some embodiments, at least one of the chemicaland/or biological reagents can be present in the sample container beforea fluid sample is added to the sample container. For example, blood canbe collected into a blood collection tube such as VACUTAINER®, which hasalready contained heparin or citrate. Examples of the chemical and/orbiological reagents can include, without limitations, surfactants anddetergents, salts, cell lysing reagents, anticoagulants, degradativeenzymes (e.g., proteases, lipases, nucleases, collagenases, cellulases,amylases), and solvents such as buffer solutions.

In some embodiments, a fluid sample can comprise certain cells isolatedfrom a biological sample and resuspended in a buffered solution orculture medium. For example, fractions of blood or platelets can beisolated from a blood sample and resuspended in a buffered solution orculture medium. As used herein, the term “culture media” refers to amedium for maintaining a tissue, an organism, or a cell population, orrefers to a medium for culturing a tissue, an organism, or a cellpopulation, which contains nutrients that maintain viability of thetissue, organism, or cell population, and support proliferation andgrowth.

E. Example 1. Development of a Microfluidic Platelet Function AssessmentInteraction (μPFA) Devices/Assays

In one aspect, microfluidic platelet function assessment (μPFA) devicescomprising or consisting essentially of at least one layer or multiplelayers (e.g., at least two layers or more) of microfluidic chambers,separated by one or more porous membranes, were developed. The porousmembranes can act as a biomimetic interstitium and comprise living orfixed cells adhered thereto. In some embodiments, at least one chambercan have a monolayer of endothelial cells (normal or cytokine activated;live or fixed) and/or extracellular matrix proteins, such as collagen,and the chamber itself can be perfused with whole blood (a biomimeticblood vessel—“vascular chamber”) (FIG. 54A). The monolayer ofendothelial cells and/or extracellular matrix proteins can be adhered tothe side of the membrane facing the chamber and/or at least one or moreof the chamber fluid-contact surfaces (e.g., a portion of the chamberfluid-contact surface or entire chamber fluid-contact surface). In someembodiments, the vascular chamber can comprise or consist essentially ofa microchannel dimensioned to be comparable to an arterial blood vessel.For example, in one embodiment, the microchannel can be a rectangularmicrochannel equivalent in size to a ˜125 μm size arterial blood vessel.In one embodiment, the microchannel can have a width of about 400 μm anda height of about 100 μm (FIGS. 54B-54C).

In some embodiments, the μPFA devices can comprise a second chamberseparating from the first chamber by a porous membrane. The secondchamber can comprise cultured cells of interest (e.g., but not limitedto astrocytes, pericytes, hepatocytes, respiratory epithelial cells, andany combinations thereof) that exhibit the functionality of a tissue ofinterest (FIG. 54D). The entire device, therefore, can represent aphysiologically relevant, three dimensional, organ system that permitsblood flow and can enable dynamic interaction of blood cells such asplatelets with the endothelium and its impact on the perivascular cells,maintained in the device.

In some embodiments, the vascular coating (e.g., the monolayer ofendothelial cells and/or extracellular matrix proteins that are incontact with flowing blood) can also mimic inflammatory endothelialconditions (e.g., endothelial dysfunction) by culturing the cells in thepresence of cytokines such as tumor necrosis factor alpha (TNF-α) (FIG.54E).

The μPFA devices described herein can be utilized at patients' bedside,e.g., for example, to analytically measure the spatiotemporal dynamicsof platelets in whole human blood at a user-designated flow rate (shearstress). In some embodiments, no more than 0.5 ml of blood is needed foreach assay using the μPFA devices described herein.

This is one of the key departure and advancement from the existinginstruments and analytical devices that are currently applied to measureplatelet function and reactivity, because the existing instruments andanalytical devices do not incorporate shear stresses, blood vesselgeometries, and interactions with inflamed endothelium or perivasculartissue.

In some embodiments, to assess platelet function using one or moreembodiments of the μPFA devices described herein, human whole blood canbe drawn in standard 3.2% sodium citrate vacutainers, e.g., byphlebotomy, and the assay can be performed within a period of time ofthe blood draw. In one embodiment, the assay can be performed within 6hours of the blood draw. The collected blood can be stored in areservoir attached to an inlet of the μPFA devices described herein andintroduced into the “vascular chamber,” for example, via a syringe pump,at a designated flow rate (shear stress) (FIG. 54A). The blood can berecalcified, e.g., using 100 mM calcium chloride and/or 75 mM magnesiumchloride solution (100 μL/mL blood), after introduced into the “vascularchamber” of the device. In some embodiments, the blood can berecalcified about 2 minutes after the microfluidic experiments begin.

In some embodiments, platelets in a blood sample can be fluorescentlylabeled. In some embodiments, CD41 conjugated antibody can be used tolabel platelets. Imaging can be automated to acquire and stitch multipleimage tiles (e.g., about 5-10 image tiles) along the length of thechamber every certain period of time (e.g., every 15 or 30 seconds).Image processing and analysis can be done using any art-recognizedprograms, e.g., but not limited to, Matlab, ImageJ and MSExcel.

For measurement of the spatiotemporal dynamics of platelet interaction,mathematical algorithms that quantitate the dynamical interactionsbetween the platelets and the fixed endothelial cell monolayer (e.g.,cytokine stimulated fixed endothelial cell monolayer) were developed asdescribed in Example 1. Characteristic spatial and temporal indices ofplatelet dynamics computed based on acquired images of plateletinteraction in the devices can be patient-specific, e.g., whenpatient-specific blood sample and/or patient-specific endothelial cellsare used in the devices described herein. Accordingly, in someembodiments, these spatial and temporal indices of platelet dynamics canbe used as prognostic or diagnostic markers of platelet-related diseasesand/or to help in modulating antiplatelet therapy to prevent recurrentthrombosis or bleeding. In some embodiments, these indices can be usedas quantitative markers of drug efficacy or toxicity, when the devicesdescribed herein are used for drug or small molecule screening (e.g.,novel drug compounds). Thus, in some embodiments, these indices can beused to determine drug toxicology.

F. Example 2. Exemplary Quantitative Algorithms for Computing PlateletAdhesion Kinetics and/or Dynamics

This Example describes exemplary embodiments of mathematical algorithmsthat each can used alone or in combination to quantitate the dynamicinteractions between platelets and one or more natural and/or artificialsurfaces. In some embodiments, the natural and/or artificial surface canform a wall portion or entire wall on all sides of the “vascularchamber” of the μPFA devices as described herein. The natural and/orartificial surface can be coated with extracellular matrix molecules(e.g., collagen), cultured with an endothelial cell monolayer (e.g.,live or fixed cells with or without cytokine stimulation), and/or a baresurface of the device (e.g., PDMS).

Adhesion was almost completely suppressed on human umbilical veinendothelial cell (HUVEC)-coated chambers or channels, indicating thatplatelets are not adhering and are mimicking transport in vivo inside ahealthy blood vessel (FIG. 55). However, TNF-α stimulated HUVECs (TNF+;TNF++) caused an increase in platelet adhesion and the HUVECs treatedwith a higher concentration of TNF-α caused a higher increase inplatelet adhesion as compared to the HUVECs treated with a lower TNF-αconcentration, indicative of an active and dynamic endothelial-plateletcrosstalk under inflammatory conditions. However, these adhesion rateswere still significantly lower when compared to a collagen surface,which is one of the most potent natural platelet agonist.

G. Example 3. A Fixed Endothelialized Surface as a PhysiologicallyRelevant Activator for Platelet Function Analysis

To increase practicality and reliability of the μPFA devices so that itcan also be utilized at the patient bedside environment, such as roomtemperature, variable humidity etc. and stored for longer periods, theinventors have discovered that platelet adhesion and dynamics can besimilarly reproduced over an endothelial cell monolayer that has beenfixed, for example, with 3% formaldehyde, as compared to a live culturedlayer. Without wishing to be bound by theory, when the endothelium isfixed, it can still conserve the expression of many procoagulatoryproteins such as vWF and tissue factor, that results in a spatially andtemporally heterogeneous surface, like in vivo or live cells (FIG. 58).This novel physiologically relevant surface, heterogeneously presentingprocoagulant molecules such as vWF and tissue factor, can allow a moreaccurate and patient-specific platelet function analysis. This surfaceis significantly different from a uniform monolayer of collagen that isclassically used in existing flow chambers for analyzing plateletfunction and thrombosis. To demonstrate reliability of measuringplatelet function over this fixed endothelial surface, platelet adhesionrate was measured on fixed HUVECs (ENDO*) and kept for 1 day or 5 days.It was determined that relative to the control (no treatment with TNF-αprior to fixation), the adhesion rates of platelets were significantlyhigher when the endothelial cells were treated with TNF-α at 5 ng/mlphysiological concentration, followed by paraformaldehyde fixation(TNF+) (FIG. 59A). Yet, the fixed endothelial surface was much lessadhesive than fixed collagen surface (COL*). In addition, the TPD (FIG.59B) and SPD (FIG. 59C) analysis as described in Example 2 showedsimilar trends, that is, platelet dynamics over treated and fixedendothelium was elevated relative to fixed collagen or untreated, fixedendothelium.

While the devices stored for 5 days at 4° C. showed increased dynamicbehavior, as compared to when they were stored for only a day, thedifference relative to the controls was still significant (FIGS.59A-59C). Without wishing to be bound by theory, fixation withparaformaldehyde can be partially reversible, and/or the endothelialsurfaces can have undergone morphological change over time impacting thedynamics. As shown in FIG. 59A, longer storage time does not appear tosignificantly affect total adhesion.

H. Example 4. Platelet Analysis on an Organ-On-A-Chip Integrated withLuminal Whole Blood Transport

Using a blood-brain-barrier-on-a-chip as an example, in one embodiment,the μPFA device can comprise a first chamber and a second chamber,wherein the first chamber and the second chamber are separated by aporous membrane. The first chamber can comprise live culturedastrocytes, while the second chamber can comprise endothelial cells. Insome embodiments, at least one or all of the walls (including the sideof the membrane facing the second chamber) can be lined with anendothelial cell monolayer. The endothelial cells can be treated with orwithout a pro-inflammatory factor. In this Example, both the astrocytesand the endothelial cells were treated with TNF-α prior to exposure toblood perfusion, thus creating an inflammatoryblood-brain-barrier-on-a-chip model including whole blood transport thatcan be utilized, for example, for the study of thrombosis, plateletactivation, aggregation, platelet-endothelial and platelet-epithelialcrosstalk (FIG. 60). Platelets were observed to be mainly on the wallsof the endothelial compartment, while fibrin has formed mostly in thestatic (no shear) astrocyte compartment due to the reaction betweenblood fibrinogen and thrombin. The endothelium shown in FIG. 60 is aliving cell culture without fixation. However, in some embodiments, theendothelium can be fixed to create a blood-brain-barrier-on-a-chipmodel.

I. Example 5. A Platelet Function Assessment Microdevice forQuantitative Analysis of Dynamic Platelet Interactions with EndotheliumUnder Flow

Activation, aggregation, adhesion, translocation and embolization ofplatelet-rich thrombi are finely controlled dynamical processes thatoccur during hemostasis and thrombosis as a result of vessel wall injuryor vascular inflammation. Due to the inherent complexity in the wayplatelets interact with the vessel wall, it is challenging to study allaspects of platelet function in a comprehensive, controlled andreproducible way. In one aspect, described herein is a biomimeticmicrofluidic blood perfusion assay where large-scale, spatiotemporalfluorescence imaging and statistical algorithms are applied to measureand quantify platelet-endothelial dynamics, independent of fluorescenceintensity. The device comprises, essentially consists of, or consists ofa set of microfluidic channels in which human umbilical vein endothelialcells (HUVECs) are cultured with or without various concentrations ofone or more inflammatory cytokine, e.g., Tumor Necrosis Factor-α(TNF-α). The channels are then perfused with human whole bloodcontaining fluorescently labeled platelets at a shear rate of about 750s⁻¹. Platelet-rich thrombi form and dissociate on the endothelial cellsurface over a 15 min time course and the dynamics of plateletaggregation and thrombus embolization are quantified by analyzingtemporal and spatial variances in the fluorescent signal. This analysisrevealed a TNF-α dose-dependent increase in both the spatial andtemporal dispersion (heterogeneity) of interactions between livingplatelets and endothelium in the device.

In contrast, these spatiotemporal dynamics were absent when plateletsinteracted with healthy endothelium or a cell free, collagen-coatedsurface that is commonly used to analyze platelet activation andthrombus formation in most existing flow chambers. The device andquantitative methods described here represent a valuable tool foranalyzing platelet-endothelial interactions under pathophysiologicalconditions relevant for thrombosis research, toxicology, drug screening,and clinical diagnostics.

Platelet hyper-reactivity plays a central role in the etiology ofvarious cardiovascular diseases and vascular disorders, including acutecoronary syndrome, stroke, pulmonary embolism, and diabetes. Plateletactivation also contributes to the failure of implanted cardiovascularand extracorporeal devices, such as pumps, arterial stents andartificial valves. The important role that platelets play in multiplepathologies has resulted in the development of a wide array ofantiplatelet drugs over the last two decades. For these reasons, it hasbecome increasingly important to measure platelet function in patientsreliably and accurately in laboratories for screening, diagnosis, andmonitoring of antiplatelet therapy, as well as for predicting thrombosisor recurrent bleeding. Conventional clinical tests, such as lighttransmission aggregometry or viscoelastic platelet function analysis,have been indispensable in unraveling much of what is currently knowabout platelet biology and its contribution to thrombosis. However,these assays are limited in that they often do not incorporate relevantfluid mechanics or physiological interactions with the endothelialsurface, which are key determinants of thrombosis.

Parallel plate-flow chambers are macroscale devices that are widely usedto measure thrombus formation and platelet adhesion kinetics; however,they require large sample volumes for analysis and usually do notincorporate an endothelium. While previous studies have reportedapplication of arterial shear stresses in microfluidic devices to showthat thrombus formation, platelet adhesion and aggregation can bevisualized and measured on a variety of prothrombogenic surfaces usingsmall volumes of whole blood or plasma, these microfluidic plateletfunction assays do not permit analysis of the contributions of plateletinteractions with endothelium or a fixed endothelium that lead tocomplex dynamics in which platelets tether, adhere, aggregate, detach,and/or translocate in space and time, as they do in vivo.

J. Other Examples

In one aspect, described herein is a biomimetic platelet functionassessment device that permits robust and quantitative analysis of howendothelial inflammation affects platelet dynamics during thrombosisunder flow in vitro. The device, being able to emulate relevant featuresof an in vivo blood vessel, includes many essential mediators ofthrombosis upon stimulation (FIGS. 65A-65B). Analysis shows that theintegrated interplay between platelets, thrombi, the vessel wall,blood-borne factors and flow dynamics can be analyzed in the integratedsystem-level assay (FIG. 65C). This new microfluidic method cantherefore be used in a variety of applications relevant for thrombosisresearch and clinical practice.

Blood Samples.

Citrated human blood (Research Blood Components, Cambridge, Mass.) wasused within 5 hours of blood draw, to minimize pre-analytical effects onplatelet function. Platelets were labeled with human CD41-PE antibody(10 μl/ml, Invitrogen) directly added to the blood and incubated at roomtemperature for 10 min. When analyzing the formation of fibrin, bloodsamples were added with 15 μg/ml of fluorescently labeled fibrinogen(Alexa 488, Invitrogen). The citrated blood was recalcified 2 minutesafter the beginning of each experiment by adding 100 μl/ml of a solutioncontaining 100 mM calcium chloride and 75 mM magnesium chloride to theblood.

Cell Culture.

Human umbilical vein endothelial cells (HUVECs, Lonza) were cultured inEndothelial Growth Medium-2 (EGM-2, Lonza) and used between passages 3and 7. Before seeding HUVECs in the devices, the microchannels werepre-treated with 1% (3-aminopropyl)-trimethoxysilane inphosphate-buffered saline (PBS) for 10 min, flushed sequentially with70% ethanol in water and 100% ethanol, and then incubated at 80° C. fortwo hours before rat tail collagen I (100 μg/ml in PBS; BD Biosciences)was introduced in the channels. Some of the devices were used with thecollagen coating alone (without cells) after incubation overnight at 37°C. and flushing of the channels with EGM-2. In other studies forcomparison, HUVECs (12.5×10⁶ cells/ml) were introduced into thecollagen-coated channels and incubated for 20 min at 37° C. to promotecell attachment before a second similar HUVEC suspension was thenintroduced and the devices were incubated upside down for an additional20 min to seed the cells on the ceiling and walls of the microfluidicchannels. Channels were then flushed with EGM-2 and the devices wereincubated at 37° C. under 5% CO₂ for 24 hours to promote HUVEC monolayerformation on all exposed surfaces of the microchannel. In some cases,the medium was supplemented with one or more inflammatory cytokines(e.g., 5 or 100 ng/ml TNF-α (Sigma)) to activate the HUVEC monolayer.Fluorescence microscopy was performed on endothelium that was fixed with4% formaldehyde (Sigma) and stained with antibodies against TF (SantaCruz), vWF (Abcam), VE-Cadherin (Santa Cruz), followed bycounterstaining with phalloidin and DAPI (Invitrogen).

Microfluidic Device Design.

The microfluidic platelet function assessment device was designed tooperate at an arterial shear rate of 750 sec⁻¹ (30 dynes/cm²), forexample, by maintaining the flow rate at 30 μl/min using a syringe pump.The flow rate can vary with the channel dimensions and/or fluid propertyto maintain substantially similar ranges of the arterial shear rate. Iflower flow rates are used, red blood cell sedimentation can occur,whereas undesirably large volumes of human donor blood are required ifhigher flow rates are utilized. The channel dimensions were determinedsuch that they enable real-time optical microscopic imaging using amoderate magnification (20×, 0.4 N.A.) objective. For these practicalreasons, and to mimic the size of a small blood vessel, the microdevicecontains a microchannel that is 400 μm wide, 100 μm high, and 2 cm long(FIG. 65A); the hydraulic diameter of this channel is equivalent to a160 μm diameter circular arteriole. Six of these channels were fit on astandard (75×25 mm) glass slide, allowing high assay throughput andreplicates on the same chip for testing assay reproducibility (FIG.65B).

Engineering of an Endothelial Lining in the Microchannel.

To recapitulate physiological interactions between flowing platelets andthe endothelial surface of living microvessels, HUVECs were cultured onall four collagen-coated walls of the rectangular channel. This led tothe formation of rectangular channel lined by a continuous, confluentendothelial monolayer, as demonstrated by VE-Cadherin and F-actinstaining (FIG. 71A). When the living endothelium was stimulated with oneor more inflammatory cytokine, e.g., TNF-α, monolayer integrity wasmaintained, but ICAM-1 expression increased in a dose-dependent manner(FIG. 71B), which closely mimics endothelial activation observed duringinflammation in vivo. An increase in the endothelial expression ofprothrombogenic tissue factor (TF) and von Willebrand Factor (vWF) isalso found in a dose dependent manner (FIG. 72).

Spatiotemporal Visualization of Platelet Function in Flowing Blood.

When the device is perfused with whole blood, thrombi that are rich inplatelets and fibrin form on the surface (FIG. 65C). The spatiotemporaldynamics of fluorescent platelet activity during formation of thesethrombi on the endothelial surface was analyzed by time-lapse imaging.As flow was stable for at least 15 min, all experiments were carried outwithin the first 15 minutes. In some embodiments, blood might begin toclot in the tubing connected to the chip at later times (leading todecreased perfusion rates). It was discovered that contrary to resultsobtained with surfaces coated with thrombogenic proteins, the reactivesurface of a living activated endothelium is highly heterogeneous, asevidenced by detection of significant spatial and temporal variabilityin platelet adhesion, aggregation, translocation, and embolization (datanot shown). To analyze these dynamic changes in platelet interactionswith the surface of the endothelium, an automated imaging program wascreated that creates a 10-frame panorama, collectively covering a large(6 mm long×0.665 mm wide) region of the microchannel. Image analysis waslimited to the 200 μm central region where shear rate gradients areminimal to avoid potential boundary layer effects. This resulted in ananalytical volume of ˜0.12 μL after image cropping, which permitsanalysis of ˜24,000 platelets (mean platelet count: 200,000 per μL wholeblood) in a single composite view. The temporal resolution was fixed to30 sec (limited by the speed of the image acquisition of themicroscope), and imaging was automatically performed for 30 time stepsover 15 minutes (FIG. 66A). Analysis was focused on the 2.5-12.5 mintime points of the acquired time series, K(x,y,t), which covers theperiod of steady growth (accelerating phase) of native whole bloodclotting.

Platelet Aggregate Morphology.

Perfusion of blood through the microchannels consistently resulted information of large and stable platelet aggregates when the channels werecoated with type I collagen without endothelial cells (FIG. 66B), whichis consistent with previous in vitro studies modeling hemostasis inducedby vascular wall injury. In contrast, when the collagen-coatedmicrochannel was covered with a continuous living endothelial monolayer,very little platelet interactions and aggregate formation were observedover the course of the 15 min experiment, much as what is observed inblood flowing in a healthy human blood vessel. However, when theendothelium was pre-treated with varying doses of TNF-α, plateletadhesion and aggregation again resulted, but the morphology of theplatelet aggregates was clearly distinct from the aggregates that formedon the collagen surface (FIG. 66B). The typical size of aggregates onactivated endothelium was visibly larger and they were more sparselydistributed. Interestingly, the size, shape and organization of thethrombi that formed on the activated endothelium in this in vitro modelwere reminiscent of what has been previously observed in vivo in animalmodels. Despite these distinct qualitative observations under differentconditions, no quantitative parameters currently exist for thecomparative analysis of platelet-endothelial interactions in plateletfunction assessment microdevice. In one aspect, described herein aremethods to quantify platelet-endothelial dynamic interactions in an invitro device, e.g., a platelet function assessment microdevice describedherein.

Platelet Adhesion and Aggregation.

Platelet adhesion and aggregation events that mediate arterialthrombosis are mediated by glycoproteins and integrins that areexpressed on the surfaces of platelets and endothelial cells. In most ofthe previous studies analyzing this process, the response was measuredby quantifying the percentage of the endothelial surface that wascovered with adherent platelets, with a spatial resolution of a fewhundred microns. This analysis is typically performed by binarysegmentation of the image after setting a threshold fluorescenceintensity for each image acquired. The percentage area covered is thencalculated as the ratio of the number of labeled pixels to the size ofthe binary image and plotted against time (FIGS. 73A-73B). There are twomajor limitations of this method. First, this analysis relies onvariables that can alter platelet fluorescence (e.g., dye concentration,labeling efficiency, light intensity, diffraction, exposure time, etc.)that may vary from sample to sample, or experiment to experiment.Another significant problem is that the area averaging parameter doesnot provide any in-depth information regarding the heterogeneity of theplatelet aggregates or the variation in spatiotemporal platelet dynamics(i.e., whether the individual platelet-rich thrombi are adhering,aggregating, translocating or embolizing).

To analyze the behavior of platelet aggregates and thrombus forming oncollagen or activated endothelium directly (i.e., independently offluorescence intensity), the coefficient of variance (CV)—the ratio ofstandard deviation to the mean—of the fluorescent signal over time wascalculated, and a t-projection of the time series (K) was performed,resulting in a spatial map, M(x,y), of platelet adhesion and aggregationdynamics (FIG. 67A). The resulting spatial image can then be reprocessedusing a color map and analyzed using an intensity palette look-up tableto contrast highly active versus dormant areas (FIG. 67A). This imageintensity transformation enabled visualization of dynamic behavior ofindividual platelet aggregates, in addition to conveying the overallpattern of platelet aggregation. For example, a uniform plateletadhesion pattern with a narrow range of temporal variance on a cell freecollagen surface was observed (FIG. 67A). When blood was flowed over ahealthy endothelium, platelets show very limited reactivity with theapical surface and therefore, the color spectrum was almost entirelyblack; however, the platelet patterns on endothelium treated TNF-α wereheterogeneous and fluctuated in a dose-dependent manner (FIG. 67A andFIG. 74).

An Aggregation Index (AI) was developed to quantitatively capturespatial variance in platelet-rich thrombus formation on collagen andendothelium. AI corresponds to the statistical inter-quartile range(IQR) (difference between the third and first quartile of CV values) foreach image M(x,y). This analysis revealed that platelet behavior on thecollagen surface was highly reactive, but uniform, and there wasnegligible adhesion or reactivity on surface of the healthy endothelium;thus both had a low AI (FIG. 67B). Further, it was found that the AI ofplatelet-rich thrombi on inflamed endothelium varied depending on thedose of TNF-α, and hence, the state of inflammation (FIG. 67B).

Thrombus Translocation and Embolization.

It was observed that in addition to platelets adhering and aggregatinginto larger platelet-rich thrombi, the thrombi themselves wouldsometimes also translocate or embolize over time. This embolization ofplatelet-rich thrombi is important from a clinical perspective, asembolisms sometimes may lead to fatal complications. Time-averagedparameters analyzed in in vitro assays, such as area fraction and AI, donot adequately capture this process. Thus, to analyze temporal plateletand thrombotic processes, such as platelet-rich thrombi translocationand embolization, parametrically using this assay, the technique ofkymography was applied. In this method, the time series K(x,y,t) isaveraged across the x-axis and transformed to create a space-time mapN(y,t), such that the horizontal spatial axis is time (t) and thevertical axis is platelet fluorescence along the length of channel (y)(FIG. 68A). On a collagen surface, platelet adhesion on the substrateincreased at a steady rate over time and there were no deviations orabrupt changes in fluorescence (FIG. 68A). Similar analysis of anunstimulated (control) endothelial surface resulted in a uniform anddark kymograph (FIG. 68A). However, the kymographs on TNF-α treatedendothelium exhibited great variation in the fluorescent signal overtime and space representing translocation or embolization ofplatelet-rich thrombi, and this behavior altered in a dose-dependentmanner.

A fluorescence-independent quantitative parameter was defined to capturethis variation in pattern, or embolization index (EI), which correspondsto the statistical coefficient of variance (CV) of the image N(x,y).Similar to the results obtained with the AI, surfaces coated withcollagen or quiescent endothelium exhibited a low EI, as the plateletbehavior was either uniformly highly reactive or negligible,respectively (FIG. 68B). This analysis also showed that the EI ofplatelets on inflamed endothelium increased with increasing doses of oneor more inflammatory cytokine, e.g., TNF-α, and thus this parameter wasable to capture the processes of translocation and embolization in thismodel of an inflamed vessel.

In one aspect, described herein is a new microdevice with integratedanalytical methods that represent a novel in vitro tool forquantitatively assessing the dynamic functions of platelet and thrombusinteractions with living endothelium under flow. This assay can beutilized in biomedical research or clinical settings because of itsease-of-use, small sample size, automated analysis and high informationcontent. The novel analytical methods described herein have severaladvantages relative to the existing microfluidic thrombosis and plateletanalysis models. First, endothelial cells are an integrated component ofthe assay, which allows one to study the interplay of endothelialdysfunction and blood-derived factors in causing thrombosis or bleeding.This advantage is clearly demonstrated by the finding presented hereinthat TNF-α treatment produces dose-dependent effects on several aspectsof platelet dynamics when endothelium is present. The other mainadvantage of the methods described herein is that it simultaneouslypermits stochastic analysis of relevant parameters of platelet dynamicsat a large scale and by enabling high resolution visualization ofcellular responses at the single platelet level. Moreover, the abilityto quantify and compare various parameters relating to platelet function(adhesion, aggregation, translocation and embolization of platelet-richthrombi), while also carrying out morphological observations, enabledclear comparison between the effects of the different biomimeticsurfaces. The working principle of this microfluidic assay and/ormethods described herein is also flexible, in that the methods describedherein can be integrated with a variety of other biomedical assays andin vitro disease models. For example, the methods or assays describedherein can be easily combined with fibrin analysis, by introducingfluorescent fibrinogen along with labeled platelets (FIG. 65C). In someembodiments, the methods and/or devices described herein can also beintegrated with organ-on-a-chip technology to study the effects ofparenchymal tissue damage, organ inflammation, and vascular (orperivascular) tissue dysfunction on platelet dynamics and thrombosis invitro in a comprehensive fashion.

In addition, a standardized device can permit this assay to be used toevaluate patient samples in clinical diagnostic settings. This abilityto assess the full spectrum of platelet function enables a more informedrisk assessment for thrombosis in at-risk disease populations. Forexample, the fluorescence microscopic analysis can be replaced withother imaging modalities, such as wide-field holography or impedancespectroscopy.

K. Example 6. Whole Blood Platelet Analysis on a Chemically PreservedBioactive Endothelium Inside a Microfluidic Device

Thrombosis depends on blood interacting with an inflamed vascularendothelium under flow, but it is impractical to incorporate livingendothelial cells in platelet function diagnostic devices used inlaboratories or at the bedside. In one aspect, described herein is amicrofluidic device lined by a non-living, fixed, stimulated endotheliumthat supports formation of platelet-rich thrombi as blood flows throughits channels. The clinical value of chemopreserved endothelializeddevices is demonstrated herein, e.g., by showing that they can be usedto monitor antiplatelet therapy in cardiac patients.

Mutual signaling between an inflamed endothelium and activated plateletsis commonly recognized as the cause of disturbances in hemostasis,platelet aggregation and resulting thrombotic disorders in variousdiseases, yet no reliable diagnostic assays exist that can measure theeffects of cross-talk between platelets and an inflamed vessel wall.Microfluidic devices that incorporate microchannels with a physiologicalrelevant size that are lined by living inflamed endothelium exposed toflowing blood can be used to study thrombosis in vitro. However, it isnot practical to incorporate living endothelial cells in clinicaldiagnostic devices given problems associated with culture stability,robustness, standardization, storage, and shipping.

To this end, the inventors recapitulated platelet-endothelial crosstalkby culturing human umbilical vein endothelial cells (HUVECs) on all fourwalls of a type I collagen coated rectangular channel (400×100 μm),which led to formation of a rectangular tube lined by a continuous,confluent endothelial monolayer (FIGS. 69A-69B), as described in Example5. The monolayers were either left untreated or were treated for 18hours with varying doses of the pro-inflammatory cytokine tumor necrosisfactor-alpha (TNF-α). After the endothelial monolayer was formed andinflammatory treatment was complete, these endothelium-lined deviceswere chemically preserved, for example, by fixing them with 4%formaldehyde diluted in phosphate buffered saline (PBS) for 15 minutes,at room temperature (FIGS. 69A-69B). After fixation, the devices wererinsed three times with PBS and then they were stored at 4° C. for 24-36hours before use. Citrated human whole blood with fluorescently taggedplatelets was perfused through these fixed endothelium-linedmicrochannels at a shear rate of 750 sec⁻¹, using less than 500 μl ofblood per assay (FIG. 69C). After 2 minutes, the blood was supplementedwith calcium (CaCl₂) and magnesium (MgCl₂) to initiate physiologicalblood clotting, which was analyzed for 2.5-12.5 min. Plateletaccumulation was measured as the percentage area occupied by theplatelets in the central 200 μm of the channel width (FIG. 75).

Adding increasing doses of one or more inflammatory cytokine, e.g.,TNF-α, to the endothelium prior to fixation resulted in a dose-dependentincrease in surface coverage of platelet-rich thrombi (FIG. 69D). Themorphology of these thrombi was distinctly different from the plateletaggregates that formed on collagen-coated device, which mimickedplatelet aggregation and adhesion that occurs on the surface of theliving endothelium during inflammation as in vivo. In contrast, therewas virtually no induction of platelet-rich thrombi formation on fixedquiescent endothelium (FIG. 69D). In addition, no significant differencein platelet accumulation was observed between a living andchemopreserved endothelium, at all the tested doses of TNF-α, thusshowing that the synthesized cellular surface retains key pro-thromboticcharacteristics after fixation (FIG. 69D, n=4). It was also shown thatthe platelet-rich thrombi that formed on the chemopreserved endothelialsurface were morphologically larger than the aggregates that formed on acollagen surface, but similar to a living endothelium (FIG. 76).Moreover, thrombi on this bioactive surface were rich in fibrin, whichalso showed that their formation was dependent on an active coagulationcascade (FIG. 76). This activity was further substantiated when it wasfound that the endothelium treated with a low dose (5 ng/ml) TNF-αexpressed higher levels of prothrombotic tissue factor (TF) and vonWillebrand Factor (vWF) than untreated, after fixation (FIG. 69E).

This data led to the investigation of whether an assay, containing afixed bioactive endothelial substrate, can be used to detectanti-platelet drug dose effects, and to compare to a similar sizedcollagen-coated microchannel using the standard LTA (light transmissionaggregometry). Thus, a concentration of 5 ng/ml of TNF-α was selectedfor causing endothelial activation, which is in the pathophyisologicallyrelevant range. First, when an antiplatelet GP IIb/IIIa antagonist drug,abciximab (ReoPro), was added in the range 0-100 μg/ml (clinical range˜1-10 μg/ml) to whole blood and platelet adhesion was measured, asignificant dose-dependent platelet inhibition was found betweenuntreated and 1 μg/ml drug and between 1 μg/ml and 10 μg/ml drug (n=3,FIG. 70A). The difference between 10 μg/ml and 100 μg/ml wasinsignificant, because at these high doses, platelet inhibition wasmaximized. Surprisingly, the dose-dependent effect of abciximab onsurface coverage in a collagen-coated flow chamber had poor sensitivitywhereas abciximab-treated platelets demonstrated no plateletaggregability by LTA in response to either ADP (adenosine diphosphate)or collagen agonists (FIGS. 70B-70C). This validated that the plateletaggregation measurement on the chemopreserved endothelium provided adynamic response across a range of abciximab concentrations indicatingthat a chemopreserved endothelium can be used to monitor anti-plateletregimens in patients. This also showed that the surface conservedplatelet interactions via the GPIIb/IIIa pathway, involved in manythrombotic and vascular processes.

Whole blood of patients who underwent angiography at a cardiaccatheterization lab in the clinic are regular users of antiplateletdrugs, e.g., aspirin alone or both aspirin and clopidogrel (Table 1).

TABLE 1 Clinical characteristics of subjects tested for plateletaggregation Subject Aspirin Clopidogrel 7 + 0 8 + 0 9 + + 16 + 0 18 + +22 + 0 59 + + 61 + 0 63 + + 65 + + 84 0 0 85 + 0 106 + 0 109 + 0 113 0 0117 + 0

Thus, it was next sought to perfuse whole blood of these patients and todetermine if the assays with a chemopreserved endothelium can be used todetermine effects of antiplatelet drugs. In a subject population thatwas tested (n=11), it was found that compared to healthy donors,patients showed a significant reduction in platelet aggregation in thedevice described herein, consistent also with LTA (FIGS. 70D-70F). Also,both healthy controls and patients showed complete platelet inhibitionover an untreated endothelium but when the endothelium was stimulated,both groups showed some signs of platelet rich thrombus formation,showing that subjects may have a higher tendency to thrombosis whenvascular inflammation is present (FIG. 70D). These data also indicatethat patients on antiplatelet agents who show platelet adhesion similarto healthy donors can be advised for monitoring as they may be at riskfor a thrombotic event. These results could not be reproduced with thesame sensitivity on a collagen, unendothelized coated flow chamber (FIG.70E), demonstrating that the assay described herein is more reliable forassessing platelet reactivity in a clinical setting.

In vitro humanized disease models of thrombosis offer opportunities tosignificantly improve diagnostics and predict patient outcome, whereblood-endothelial interactions are involved. In one aspect, describedherein is a microfluidic device that contains a layer of chemicallypreserved endothelial cells that are either quiescent or pre-stimulatedwith TNF-α prior to fixation. The chemically preserved endotheliumretains its respective passive or pro-thrombotic properties as if itwere alive (FIGS. 69A-69E). It is demonstrated herein that these devicescan be used to evaluate platelet aggregation and inhibition with drugs.This technology can enhance platelet function analysis at bench orbedside.

L. Exemplary Materials And Methods

Microfluidic Device Design and Fabrication.

Microfluidic device consisted of a microchannel, 400 μm wide, 100 μmhigh and 2 cm long. It was designed using AutoCAD™ software, mastertemplates fabricated on Si (100) wafers (University Wafer Corp.) incombination with soft lithography using polydimethysiloxane (PDMS).Duffy et al. “Rapid prototyping of microfluidic systems inpoly(dimethylsiloxane). Anal. Chem. 70, 4974-4984 (1998). Sylgard 184™PDMS prepolymer (Dow Corning) was cast on the silanized master that hadthe positive relief of the channel features formed by SU8 2075photoresist (MicroChem Corp). The PDMS was then cured at 60° C. in aconvection oven for 120 minutes, peeled off the master, and bonded to aPDMS coated glass slide after treating both with oxygen plasma for 20seconds.

Microfluidic Device Pre-Treatment and Coating.

The microfluidic devices were pre-treated and coated with collagenbefore cell seeding. Devices were exposed to oxygen plasma for 30seconds, at a power of 50 Watts, using a PE-100™ plasma sterilizer(Plasma Etch, Inc. NV, USA) and then treated with 1%(3-aminopropyl)-trimethoxysilane (Sigma) in phosphate-buffered saline,PBS, for 10 minutes. After rinsing with 70% ethanol and 100% ethanol,the devices were baked at 80° C. for 2 hours. A solution of 100 μg/mltype I collagen from rat tail (Corning) in PBS was then introduced inthe channels. The devices were left overnight at 37° C. and 5% CO₂,after which they were rinsed with Endothelial Growth Medium-2, EGM-2(Lonza).

Cell Culture and Chemical Preservation.

Human umbilical vein endothelial cells, HUVEC, (mixed donor, Lonza) werekept in culture with EGM-2 and were trypsinized when confluent. Aftercentrifugation at 250 g, HUVEC were suspended at a 12.5 million cells/mlin EGM-2. The suspension was introduced into the pre-treated and coatedmicrochannels, after which the devices were incubated upside down for 20minutes. A fresh HUVEC suspension was then introduced in the channels,after which the devices were covered with EGM-2 and left at 37° C., 5%CO₂ for 8 hours to promote cell attachment and spreading on all surfacesof the channel. After incubation, the channels were rinsed with EGM-2,sometimes containing a freshly prepared solution of tumor necrosisfactor-alpha TNF-α (recombinant from E. coli, Sigma). After incubatingfor 18 to 20 hours at 37° C., 5% CO₂, a 4% formaldehyde solution (Sigma)was flushed through the channels and the devices were incubated for 15minutes at room temperature. Finally, the devices were rinsed twice withEGM-2 and then placed at 4° C. The devices were used within 24-36 hoursafter placing them at 4° C.

Fluorescent Labeling of Platelets.

Platelets labeled with human CD41-PE antibody (10 μl/ml, Invitrogen)were directly added to the blood and incubated at room temperature for10 min. The citrated blood was recalcified 2 minutes after bloodperfusion by adding 100 μl/ml of a solution containing 100 mM calciumchloride and 75 mM magnesium chloride to the blood.

Light Transmission Aggregometry (LTA).

Blood from healthy donors was treated ex vivo with abciximab or useduntreated. These samples as well as clinical blood samples from subjectswere centrifuged at 290 g for 10 min (no brake applied) to collectplatelet rich plasma (PRP). To obtain a reference solution for eachsample, PRP was centrifuged at high speed to pellet platelets (1,000 g,10 min) and collect platelet poor plasma (PPP). CaCl₂ was added to eachsample at a final concentration of 1 mM to recalcify plasma before eachrun. Cifuni et al. “CalDAG-GEFI and protein kinase C representalternative pathways leading to activation of integrin alphallbeta3 inplatelets.” Blood 112, 1696-1703 (2008). LTA has then been performed at37° C. under magnetic stirring using a Chrono-Log Corporationinstrument. Both platelet agonists, ADP (adenosine diphosphate) andcollagen, were purchased from Chrono-Log Corporation and used assuggested by the manufacturer, in the concentrations of 10 μM and 2μg/ml, respectively.

Blood Perfusion.

500 μl of whole blood was pipetted into a fluid reservoir fitted to oneend of the microchannel on one side of the microfluidic device. A pieceof medical grade tubing (30.5 mm long, 1.58 mm inner diameter; TygonS-50-HL, Saint Gobain Plastics) was fitted to the outlet port of thedevice via a barbed luer lock connector (Harvard Apparatus). The otherend of the tube was connected to a 3 ml syringe (Becton Dickinson)through which blood was withdrawn from the device by pulling (30 μl/min)using a syringe pump (PHD Ultra CP, Harvard Apparatus), thereby drivingblood flow through the microchannels (FIG. 69A). Recalcification ofblood was performed after 2 min of operation to permit calcium- andmagnesium-dependent platelet-derived thrombus formation.

VIII. First Set of Alternative Embodiments A. Embodiments A1-A9Embodiment A1

A microchannel comprising one or more surfaces, the microchannel havingliving endothelial cells on all of the microchannel surfaces.

Embodiment A2

The microchannel of embodiment A1, wherein the living endothelial cellsare human umbilical vein endothelial cells.

Embodiment A3

The microchannel of embodiment A1, wherein the surfaces are coated withat least one attachment molecule that supports adhesion of the livingendothelial cells.

Embodiment A4

The microchannel of embodiment A1, wherein the microchannel includes atop surface, a bottom surface, a first side surface, and a second sidesurface.

Embodiment A5

The microchannel of embodiment A4, wherein the bottom surface includes amembrane.

Embodiment A6

The microchannel of embodiment A1, wherein the microchannel is in fluidcommunication with an input port and an output port.

Embodiment A7

The microchannel of embodiment A1, wherein the microchannel has a widthin the range of about 50 microns to about 1,000 microns.

Embodiment A8

The microchannel of embodiment A1, wherein the microchannel has a heightin the range of about 50 microns to about 200 microns.

Embodiment A9

The microchannel of embodiment A1, wherein the microchannel includes atube lined with a continuous, confluent layer of endothelial cells.

B. Embodiments B1-B5 Embodiment B1

A device comprising: a body having a microchannel therein, themicrochannel including one or more surfaces, the microchannel includingliving endothelial cells on all of the microchannel surfaces.

Embodiment B2

The device of embodiment B1, wherein the microchannel includes a topsurface, a bottom surface, a first side surface, and a second sidesurface.

Embodiment B3

The device of embodiment B2, wherein the bottom surface includes amembrane.

Embodiment B4

The device of embodiment B3, wherein the membrane is at least partiallyporous.

Embodiment B5

The device of embodiment B1, further comprising an input port and anoutput port, the ports being in fluidic communication with themicrochannel.

C. Embodiments C1-C3 Embodiment C1

A system comprising: a) a microchannel having one or more surfaces; b)living endothelial cells on all of the surfaces; and (c) fluid movingthrough the microchannel.

Embodiment C2

The system of embodiment C1, wherein the fluid includes whole blood thatcontacts the endothelial cells without clotting.

Embodiment C3

The system of embodiment C1, wherein the fluid includes platelets, theplatelets being in contact with the endothelial cells without clotting.

D. Embodiments D1-D8 Embodiment D1

A method comprising:

1) providing

-   -   a) a microchannel with one or more surfaces, and    -   b) living endothelial cells on all of the surfaces; and

2) introducing fluid into the microchannel.

Embodiment D2

The method of embodiment D1, wherein the living endothelial cells arehuman umbilical vein endothelial cells.

Embodiment D3

The method of embodiment D1, wherein the fluid is selected from a groupconsisting of a blood sample, a serum sample, a plasma sample, a lipidsolution, a nutrient medium, or a combination of two or more thereof.

Embodiment D4

The method of embodiment D1, wherein the fluid includes whole blood thatcontacts the endothelial cells without clotting.

Embodiment D5

The method of embodiment D1, wherein the fluid includes platelets, theplatelets contacting the endothelial cells without clotting.

Embodiment D6

The method of embodiment D1, further comprising, prior to step 2),exposing the living endothelial cells to a pro-inflammatory cytokine.

Embodiment D7

The method of embodiment D6, wherein the fluid includes whole blood thatcontacts the endothelial ceils under conditions such that aplatelet-rich thrombus forms.

Embodiment D8

The method of embodiment D6, wherein the fluid includes platelets, theplatelets clotting upon contacting the endothelial cells.

E. Embodiments E1-E8 Embodiment E1

A method comprising:

1) providing

-   -   a) a microchannel having one or more surfaces, and    -   b) fixed endothelial cells on all of the surfaces; and

2) introducing fluid into the microchannel.

Embodiment E2

The method of embodiment E1, wherein the fixed endothelial cells arehuman umbilical vein endothelial cells.

Embodiment E3

The method of embodiment E1, wherein the fluid is selected from a groupconsisting of a blood sample, a serum sample, a plasma sample, a lipidsolution, a nutrient medium, and a combination of two or more thereof.

Embodiment E4

The method of embodiment E1, wherein the fluid includes whole blood thatcontacts the endothelial cells without clotting.

Embodiment E5

The method of embodiment E1, wherein the fluid includes platelets, theplatelets contacting the endothelial cells without clotting.

Embodiment E6

The method of embodiment E1, wherein the endothelial cells arephysically fixed by at least one of drying and dehydration.

Embodiment E7

The method of embodiment E1, wherein the endothelial cells are fixed byat least one of exposing to air, washing with alcohol, acetone, or asolvent that removes at least one of water and lipids.

Embodiment E8

The method of embodiment E1, wherein the endothelial cells are fixedwith a chemical fixative.

IX. Second Set of Alternative Embodiments A. Embodiments A1-A10Embodiment A1

A method of testing a drug, the method comprising:

a. providing a fluid sample of a subject, the fluid sample includingplatelets;b. adding a drug to a portion of the fluid sample to create a testsample;c. flowing the test sample through a microchannel of a microfluidicdevice, the microchannel including one or more surfaces having anendothelial cell monolayer thereon, the endothelial cells being in astimulated state;d. detecting interaction between platelets in the test sample and theendothelial cells;e. comparing the level of interaction of step d) with that of a control;andf. determining whether the drug interfered with a platelet function.

Embodiment A2

The method of embodiment A1, wherein the control includes the fluidsample of the subject without the drug.

Embodiment A3

The method of embodiment A1, wherein the endothelial cells arestimulated with a cytokine.

Embodiment A4

The method of embodiment A3, wherein the cytokine is TNF-α.

Embodiment A5

The method of embodiment A1, wherein the drug is an antiplatelet GPIIb/IIIa antagonist.

Embodiment A6

The method of embodiment A1, wherein the drug is an antibody.

Embodiment A7

The method of embodiment A1, wherein the drug is abciximab.

Embodiment A8

The method of embodiment A1, wherein the microchannel has a top surface,a bottom surface, a first side surface, and a second side surface.

Embodiment A9

The method of embodiment A8, wherein the microchannel includes livingendothelial cells on all of the microchannel surfaces.

Embodiment A10

The method of embodiment A8, wherein the microchannel includes fixedendothelial cells on all of the microchannel surfaces.

X. Third Set of Alternative Embodiments A. Embodiments A1-A14 EmbodimentA1

A method of determining if a subject is at risk, or has a disease ordisorder, induced by platelet dysfunction, the method comprising:

a. flowing a fluid sample of the subject including platelets over asurface having an endothelial cell monolayer thereon;b. detecting interaction between platelets in the fluid sample and theendothelial cells;c. comparing the level of interaction of step b) with that of a control;andd. identifying the subject to be at risk, or have the disease ordisorder, induced by platelet dysfunction when the platelet interactionis higher than the control.

Embodiment A2

The method of embodiment A1, wherein the subject is at increased riskfor thrombosis.

Embodiment A3

The method of embodiment A2, further comprising selecting an appropriatetherapy and administering the therapy to the subject.

Embodiment A4

The method of embodiment A3, wherein the therapy is anti-platelettherapy.

Embodiment A5

The method of embodiment A3, wherein the therapy is anti-inflammationtherapy.

Embodiment A6

The method of embodiment A1, wherein the disease or disorder induced byplatelet dysfunction is an inflammatory vascular disease.

Embodiment A7

The method of embodiment A1, wherein the disease or disorder induced byplatelet dysfunction is a cardiovascular disorder.

Embodiment A8

The method of embodiment A1, wherein the surface having an endothelialcell monolayer is the surface of a microchannel of a microfluidicdevice, the device including a body having a microchannel therein.

Embodiment A9

The method of embodiment A8, wherein the microchannel includes a topsurface, a bottom surface, a first side surface, and a second sidesurface.

Embodiment A10

The method of embodiment A9, wherein the microchannel includes livingendothelial cells on all of the microchannel surfaces.

Embodiment A11

The method of embodiment A9, wherein the microchannel includes fixedendothelial cells on all of the microchannel surfaces.

Embodiment A12

The method of embodiment A9, wherein the bottom surface includes amembrane.

Embodiment A13

The method of embodiment A12, wherein the membrane is at least partiallyporous.

Embodiment A14

The method of embodiment A8, wherein the device further includes aninput port and an output port, the ports being in fluidic communicationwith the microchannel.

B. Embodiments B1-B11 Embodiment B1

A method of determining if a subject is at risk or has a disease ordisorder induced by platelet dysfunction, the method comprising:

a. flowing a fluid sample of the subject having platelets through amicrochannel of a microfluidic device, the microchannel having one ormore surfaces with an endothelial cell monolayer thereon;

b. detecting interaction between platelets in the fluid sample and theendothelial cells;

c. comparing the level of interaction of step b) with that of a control;and

d. identifying the subject to be at risk or have the disease or disorderinduced by platelet dysfunction when the platelet interaction is higherthan the control.

Embodiment B2

The method of embodiment B1, wherein the subject is at increased riskfor thrombosis.

Embodiment B3

The method of embodiment B2, further comprising selecting an appropriatetherapy and administering the therapy to the subject.

Embodiment B4

The method of embodiment B3, wherein the therapy is anti-platelettherapy.

Embodiment B5

The method of embodiment B3, wherein the therapy is anti-inflammationtherapy.

Embodiment B6

The method of embodiment B1, wherein the disease or disorder induced byplatelet dysfunction is an inflammatory vascular disease.

Embodiment B7

The method of embodiment B1, wherein the disease or disorder induced byplatelet dysfunction is cardiovascular disorder.

Embodiment B8

The method of embodiment B1, wherein the microchannel includes a topsurface, a bottom surface, a first side surface, and a second sidesurface.

Embodiment B9

The method of embodiment B8, wherein the microchannel includes livingendothelial cells on all of the microchannel surfaces.

Embodiment B10

The method of embodiment B8, wherein the microchannel includes fixedendothelial cells on all of the microchannel surfaces.

Embodiment B11

The method of embodiment B8, wherein the bottom surface includes amembrane.

C. Embodiments C1-C4 Embodiment C1

A method of determining if a subject on an antiplatelet agent is at riskfor a thrombotic event, the method comprising:

a. flowing a fluid sample of the subject having platelets through amicrochannel of a microfluidic device, the microchannel having one ormore surfaces with an endothelial cell monolayer thereon, theendothelial cells being in a stimulated state;

b. detecting interaction between platelets in the fluid sample and theendothelial cells;

c. comparing the level of interaction of step b) with that of a healthycontrol; and

d. identifying the subject to be at risk of a thrombotic event when theplatelet interaction is higher than the healthy control.

Embodiment C2

The method of embodiment C1, wherein the platelet interaction includesplatelet adhesion.

Embodiment C3

The method of embodiment C1, wherein the antiplatelet agent is aspirin.

Embodiment C4

The method of embodiment C1, wherein the antiplatelet agent isClopidogrel.

XI. Fourth Set of Alternative Embodiments A. Embodiments A1-A40Embodiment A1

A method of determining a platelet function, the method comprising:

a. flowing a fluid sample over a surface having a fixed endothelial cellmonolayer thereon; and

b. in response to detecting interaction between platelets in the fluidsample and the fixed endothelial cell monolayer, determining a functionof the platelets in the fluid sample.

Embodiment A2

The method of embodiment A1, wherein the fixed endothelial cellmonolayer is derived from at least one of (i) fixing an endothelial cellextract and (ii) endothelial cell-associated proteins that are adheredto the surface.

Embodiment A3

The method of embodiment A2, wherein the endothelial cell-associatedproteins include at least one of a procoagulatory protein and ananti-coagulatory protein.

Embodiment A4

The method of embodiment A1, wherein the fluid sample is selected from agroup consisting of a blood sample, a serum sample, a plasma sample, alipid solution, a nutrient medium, and a combination of two or morethereof.

Embodiment A5

The method of embodiment A1, wherein the surface is a surface of amicrochannel.

Embodiment A6

The method of embodiment A1, wherein the surface is a surface of amembrane.

Embodiment A7

The method of embodiment A6, wherein the membrane is configured toseparate a first microchannel and a second microchannel in amicrofluidic device.

Embodiment A8

The method of embodiment A7, wherein the microfluidic device is anorgan-on-chip device.

Embodiment A9

The method of embodiment A7, wherein a first surface of the membranefacing the first microchannel includes the fixed endothelial cellmonolayer thereon, and a second surface of the membrane facing thesecond microchannel includes tissue-specific cells adhered thereon.

Embodiment A10

The method of embodiment A1, wherein the fixed endothelial cellmonolayer is derived from fixing an endothelial cell monolayer that hasbeen grown on the surface for a period of time.

Embodiment A11

The method of embodiment A10, wherein the endothelial cell monolayer isphysically fixed by at least one of drying and dehydration.

Embodiment A12

The method of embodiment A10, wherein the endothelial cell monolayer isphysically fixed by at least one of exposing to air and washing with atleast one of alcohol, acetone, and a solvent that removes at least oneof water and lipids.

Embodiment A13

The method of embodiment A1, wherein the endothelial cell monolayer isfixed with a chemical fixative.

Embodiment A14

The method of embodiment A13, wherein the chemical fixative is selectedfrom the group consisting of formaldehyde, paraformaldehyde, formalin,glutaraldehyde, mercuric chloride-based fixatives, precipitatingfixatives, dimethyl suberimidate (DMS), Bouin's fixative, and acombination of two or more thereof, the mercuric chloride-basedfixatives including Helly and Zenker's solution, the precipitatingfixatives including at least one of ethanol, methanol, and acetone.

Embodiment A15

The method of embodiment A10, wherein the endothelial cell monolayer isfixed with a decellularization solvent that stabilizes surface membraneprotein configuration and a cytoskeleton of a cell.

Embodiment A16

The method of embodiment A15, wherein the decellularization solventincludes an aqueous solution having at least one of a detergent and ahigh pH solution.

Embodiment A17

The method of embodiment A1, wherein endothelial cells of the fixedendothelial cell monolayer are derived from a subject.

Embodiment A18

The method of embodiment A1, wherein endothelial cells of the fixedendothelial cell monolayer are differentiated from subject-specificpluripotent stem cells.

Embodiment A19

The method of embodiment A1, wherein the fixed endothelial cellmonolayer is derived from healthy cells.

Embodiment A20

The method of embodiment A1, wherein the fixed endothelial cellmonolayer is derived from diseased cells.

Embodiment A21

The method of embodiment A20, wherein the diseased cells are generatedby contacting healthy endothelial cells with an inflammation-inducingagent prior to the treatment with a fixative.

Embodiment A22

The method of embodiment A21, wherein the inflammation-inducing agentincludes at least one of a physical stimulus, a chemical agent, abiological agent, a molecular agent, or a combination of two or morethereof.

Embodiment A23

The method of embodiment A20, wherein the diseased cells are derivedfrom a subject diagnosed with a disease.

Embodiment A24

The method of embodiment A1, further comprising, when the fluid sampleincludes a blood sample, removing red blood cells from the blood sampleprior to flowing the blood sample over the surface.

Embodiment A25

The method of embodiment A1, wherein the detecting includes measuring atleast one of temporal and spatial interaction dynamics of the plateletsin the fluid sample.

Embodiment A26

The method of embodiment A25, wherein the interaction dynamics of theplatelets includes at least one of binding dynamics of the platelets tothe fixed endothelial cell monolayer, binding dynamics of the plateletsto each other, and a combination thereof.

Embodiment A27

The method of embodiment A1, wherein the platelets in the blood sampleare label-free.

Embodiment A28

The method of embodiment A1, wherein the platelets in the blood sampleare labeled with a detectable label.

Embodiment A29

The method of embodiment A28, wherein the detectable label is afluorescent label.

Embodiment A30

The method of embodiment A1, wherein the detecting is performed by animaging-based method.

Embodiment A31

The method of embodiment A30, wherein the imaging-based method includestime-lapse microscopy.

Embodiment A32

The method of embodiment A1, wherein the detecting is performed by atleast one of a wide-field holography device, an impedance spectroscopydevice, a flow sensor, a pressure sensor, and a combination of two ormore thereof.

Embodiment A33

The method of embodiment A1, wherein the surface has been stored at roomtemperature for a period of time prior to the flowing of the fluidsample.

Embodiment A34

The method of embodiment A1, wherein the surface has been stored at atemperature of about 4° C. or lower for a period of time prior to theflowing of the fluid sample.

Embodiment A35

The method of embodiment A33, wherein the period of time is in the rangeof about 1 day to about 5 days.

Embodiment A36

The method of embodiment A1, wherein the fluid sample is flowed at aphysiological shear rate or a pathological shear rate.

Embodiment A37

The method of embodiment A1, wherein the fluid sample is flowed at ashear rate of about 50 sec⁻¹ to about 10,000 sec⁻¹.

Embodiment A38

The method of embodiment A1, wherein the fixed endothelial cellmonolayer and the fluid sample are derived from the same subject.

Embodiment A39

The method of embodiment A1, wherein the fixed endothelial cellmonolayer and the fluid sample are derived from different sources.

Embodiment A40

The method of embodiment A1, wherein the fluid sample includes at leastone of calcium ions and magnesium ions.

B. Embodiments B1-B2 Embodiment B1

A system for determining temporal dynamics of platelets in a fluidsample, the system comprising:

a. a solid substrate having a surface with a fixed endothelial cellmonolayer thereon;

b. a detection module configured to receive the solid substrate and todetect spatial and temporal interaction of platelets in a fluid samplewith the surface when the fluid sample is flowed over the surface alonga flow axis; and

c. a computer system for computing platelet dynamics, the computersystem including one or more processors and a memory to store one ormore programs, the one or more programs including instructions for:

-   -   i. storing time-lapse data of detectable signals collected from        the detection module, wherein the detectable signals represent        the spatial and temporal interaction of the platelets with the        surface,    -   ii. generating a kymograph from at least a portion of the stored        time-lapse data, wherein the time axis of the kymograph        indicates at least a portion of the time-lapse duration, and the        space axis of the kymograph indicates the detectable signals        along the flow axis,    -   iii. computing, based on the generated kymograph, a rate of        fluctuation in coefficient of variation (CV) of the detectable        signals to generate a temporal platelet dynamics index, and    -   iv. determining the presence of reactive platelets in the fluid        sample when the temporal platelet dynamics index is higher than        a temporal control value, or determining the absence of reactive        platelets in the fluid sample when the temporal platelet        dynamics index is no more than the temporal control value; and

d. a display module for displaying a content based in part on thecomputed output from the computer system, wherein the content includes asignal indicative of at least one of the presence of reactive plateletsand platelet aggregation in the fluid sample, or a signal indicative ofat least one of the absence of reactive platelets and plateletaggregation in the fluid sample.

Embodiment B2

The system of embodiment B1, wherein the detectable signals are averagedacross the width of the surface, transverse to the flow axis, prior tostacking to generate the kymograph.

C. Embodiments C1-C8 Embodiment C1

A system for determining spatial dynamics of platelets in a fluidsample, the system comprising:

a. a solid substrate having a surface with a fixed endothelial cellmonolayer thereon;

b. a detection module configured to receive the solid substrate and todetect spatial and temporal interaction of platelets in a fluid samplewith the surface when the fluid sample is flowed over the surface alonga flow axis;

c. a computer system for computing platelet dynamics, the computersystem including one or more processors and a memory to store one ormore programs, the one or more programs comprising instructions for:

-   -   i. storing time-lapse data of detectable signals collected from        the detection module, wherein the time-lapse data represents the        spatial and temporal interaction of the platelets with the        surface,    -   ii. generating, from at least a portion of the stored time-lapse        data, a spatial map of temporal variances of the detectable        signals, wherein each pixel of the spatial map corresponds to a        time-averaged coefficient of variation (CV) of the detectable        signals,    -   iii. computing, based on the generated spatial map, an        inter-quartile range (IQR) of the map to generate a spatial        platelet dynamics index, and    -   iv. determining the presence of platelet aggregation in the        fluid sample when the spatial platelet dynamics index is higher        than a spatial control value; or determining the absence of        platelet aggregation in the fluid sample when the spatial        platelet dynamics index is no more than the spatial control        value; and

d. a display module for displaying a content based in part on thecomputed output from the computer system, wherein the content includes asignal indicative of at least one of the presence of reactive plateletsand platelet aggregation in the fluid sample, or a signal indicative ofat least one of the absence of reactive platelets and plateletaggregation in the fluid sample.

Embodiment C2

The system of embodiment C1, wherein the time-lapse data is presented inthe form of images.

Embodiment C3

The system of embodiment C2, wherein the detection module includes animaging-based device.

Embodiment C4

The system of embodiment C3, wherein the imaging-based device includes amicroscope or a microscope blade.

Embodiment C5

The system of embodiment C1, wherein the display module is selected froma group consisting of a computer display, a screen, a monitor, aphysical printout, and a storage device, the content being selected froma group consisting of an email, a text message, a website, and storedinformation on the storage device.

Embodiment C6

The system of embodiment C1, wherein the one or more programs includeinstructions for determining platelet reactivity based on a linear ornon-linear function having the spatial and temporal dynamic parameters.

Embodiment C7

The system of embodiment C1, wherein the one or more programs furtherinclude instructions for computing area-averaged platelet adhesion overat least a portion of the surface.

Embodiment C8

The system of embodiment C1, wherein the fluid sample includes a bloodsample.

D. Embodiments D1-D7 Embodiment D1

A method of determining if a subject is at risk or has a disease ordisorder induced by platelet dysfunction, the method comprising:

a. flowing a fluid sample of the subject over a surface with a fixedendothelial cell monolayer thereon;

b. detecting interaction between platelets in the fluid sample and thesurface; and

c. identifying the subject to be

-   -   at risk or have the disease or disorder induced by platelet        dysfunction when the platelet interaction is higher than a        control, or    -   less likely to have a disease or disorder induced by platelet        dysfunction when the platelet interaction is no more than the        control.

Embodiment D2

The method of embodiment D1, wherein the method is implemented in acomputer system having one or more processors and a memory storing oneor more programs for execution by the one or more processors, the one ormore programs including instructions for:

i. generating a kymograph from at least a portion of time-lapse data ofdetectable signals representing spatial and temporal interaction of theplatelets with the surface, wherein the time axis of the kymographindicates at least a portion of the time-lapse duration, and the spaceaxis of the kymograph indicates the detectable signals along a flowaxis;

ii. computing, based on the generated kymograph, a rate of fluctuationin coefficient of variation (CV) of the detectable signals to generate atemporal platelet dynamics index; and

iii. determining

-   -   the presence of reactive platelets in the fluid sample when the        temporal platelet dynamics index is higher than a temporal        control value, thereby identifying the subject to be at risk or        have the disease or disorder induced by platelet dysfunction, or    -   the absence of reactive platelets in the fluid sample when the        temporal platelet dynamics index is no more than the temporal        control value, thereby identifying the subject to be less likely        to have a disease or disorder induced by platelet dysfunction.

Embodiment D3

The method of embodiment D1, wherein the method is implemented in acomputer system having one or more processors and a memory storing oneor more programs for execution by the one or more processors, the one ormore programs including instructions for:

i. generating, from at least a portion of time-lapse data of detectablesignals representing spatial and temporal interaction of the plateletswith the surface, a spatial map of temporal variances of the detectablesignals, wherein each pixel of the spatial map corresponds to atime-averaged coefficient of variation (CV);

ii. computing, based on the generated spatial map, an inter-quartilerange (IQR) of the map to generate a spatial platelet dynamics index;and

iii. determining

-   -   the presence of platelet aggregation in the fluid sample when        the spatial platelet dynamics index is higher than a spatial        control value, thereby identifying the subject to be at risk or        have the disease or disorder induced by platelet dysfunction, or    -   the absence of platelet aggregation in the fluid sample when the        spatial platelet dynamics index is no more than the spatial        control value, thereby identifying the subject to be less likely        to have a disease or disorder induced by platelet dysfunction.

Embodiment D4

The method of embodiment D1, wherein the fixed endothelial cellmonolayer is subject-specific.

Embodiment D5

The method of embodiment D1, wherein the disease or disorder induced byplatelet dysfunction is selected from a group consisting of thrombosis,an inflammatory vascular disease, a cardiovascular disorder,vasculopathies, and a combination of two or more thereof, theinflammatory vascular disease including sepsis or rheumatoid arthritis,the cardiovascular disorder including acute coronary syndromes, stroke,or diabetes mellitus, the vasculopathies including malaria ordisseminated intravascular coagulation.

Embodiment D6

The method of embodiment D1, wherein the fluid sample includes a bloodsample.

Embodiment D7 The system of embodiment D1, wherein the fluid sampleincludes a blood sample.

E. Embodiments E1-E10 Embodiment E1

A composition comprising:

a. a solid substrate having a surface with a fixed endothelial cellmonolayer thereon; and

b. a fluid sample having platelets in contact with the surface.

Embodiment E2

The composition of embodiment E1, wherein the fluid sample includes ablood sample.

Embodiment E3

The composition of embodiment E1, wherein the fixed endothelial cellmonolayer is derived from fixing an endothelial cell monolayer that hasbeen grown on the surface for a period of time.

Embodiment E4

The composition of embodiment E3, wherein the endothelial cell monolayeris derived from fixing at least one of endothelial cell extract orendothelial cell-associated proteins that are adhered to the surface.

Embodiment E5

The composition of embodiment E1, wherein the solid substrate isselected from a group consisting of a microscopic slide, a cell culturedish, a microfluidic device, a microwell, and any combinations thereof.

Embodiment E6

The composition of embodiment E1, wherein the surface is a surface of amicrochannel.

Embodiment E7

The composition of embodiment E1, wherein the surface is a surface of amembrane.

Embodiment E8

The composition of embodiment E7, wherein the membrane is configured toseparate a first microchannel and a second microchannel in amicrofluidic device.

Embodiment E9

The composition of embodiment E8, wherein the microfluidic device is anorgan-on-chip device.

Embodiment E10

The composition of embodiment E8, wherein a first surface of themembrane is facing the first microchannel has the fixed endothelial cellmonolayer thereon, and a second surface of the membrane facing thesecond microchannel has tissue-specific cells adhered thereon.

Each of these embodiments and obvious variations thereof is contemplatedas falling within the spirit and scope of the claimed invention, whichis set forth in the following claims. Moreover, the present conceptsexpressly include any and all combinations and subcombinations of thepreceding elements and aspects.

1-65. (canceled)
 66. A method of making microfluidic devices,comprising: a) providing a microfluidic device comprising a channel; b)culturing endothelial cells in the channel so as to form a lumen; c)fixing the endothelial cells; and d) shipping the device.
 67. The methodof claim 66, wherein the fixing is done with a fixative agent selectedfrom the group consisting of formaldehyde, paraformaldehyde, formalin,glutaraldehyde, mercuric chloride-based fixatives, Helly's solution,Zeneker's solution, precipitating fixatives, ethanol, methanol, acetone,dimethyl suberimidate, Bouin's fixative, a decellularization solvent, adetergent, a high pH solution, and a combination of two or more thereof.68. The method of claim 66, wherein the method further comprises thestep of storing the microfluidic device after step c).
 69. The method ofclaim 68, wherein the microfluidic device is stored at a temperaturebetween 4° C. and 10° C.
 70. The method of claim 68, wherein themicrofluidic device is stored at a temperature of about 4° C. or lower.71. The method of claim 68, wherein the microfluidic device is stored atroom temperature.
 72. The method of claim 68, wherein the microfluidicdevice is stored for 1 day or longer.
 73. The method of claim 68,wherein the microfluidic device is stored for 5 days or longer.
 74. Amethod of making microfluidic devices, comprising: a) providing amicrofluidic device comprising a channel; b) culturing endothelial cellsin the channel of each microfluidic device so as to form a lumen; c)fixing the endothelial cells; and d) storing the plurality ofmicrofluidic devices.
 75. The method of claim 74, wherein the fixing isdone with a fixative agent selected from the group consisting offormaldehyde, paraformaldehyde, formalin, glutaraldehyde, mercuricchloride-based fixatives, Helly's solution, Zeneker's solution,precipitating fixatives, ethanol, methanol, acetone, dimethylsuberimidate, Bouin's fixative, a decellularization solvent, adetergent, a high pH solution, and a combination of two or more thereof.76. The method of claim 74, wherein the microfluidic device is stored ata temperature between 4° C. and 10° C.
 77. The method of claim 74,wherein the microfluidic device is stored at a temperature of about 4°C. or lower.
 78. The method of claim 74, wherein the microfluidic deviceis stored at room temperature.
 79. The method of claim 74, wherein themicrofluidic device is stored for 1 day or longer.
 80. The method ofclaim 74, wherein the microfluidic device is stored for 5 days orlonger.